Chromobacterium formulations, compositions, metabloites and their uses

ABSTRACT

Stabilized biological pesticides comprising  Chromobacterium  species, filtrate, supernatant, extract or pesticidally active substance derived therefrom with pesticidal activity having improved shelf life due to maintenance of physical uniformity and longer insecticide activity after use due to higher resistance to degradation when exposed to sunlight are disclosed.

TECHNICAL FIELD

Provided is the use of or compositions or formulations comprising Chromobacterium species, filtrate, supernatant, extract, pesticidally active compound or metabolite derived there therefrom as an acaricide and insecticide, particularly against infestation of one or more pests belonging to the Acarina Scarabeidae, Drosophilidae, TriozidaeAphidae, Muscidae, Anthomyildae or Tenebrionidae families. Further provided are biological pesticide (also referred to as biopesticide) formulations, particularly those comprising Chromobacterium species, filtrate, supernatant, extract, metabolites or pesticidally active compounds derived therefrom, methods for producing them and methods of use as modulating pest infestation. More specifically, provided are stabilized biological pesticides having improved shelf life due to maintenance of physical uniformity and longer insecticide activity alter use due to higher resistance to degradation when exposed to sunlight.

BACKGROUND

Natural products are substances produced by microbes, plants, and other organisms. Microbial natural products offer an abundant source of chemical diversity, and there is a long history of utilizing natural products for pharmaceutical purposes. Despite the emphasis on natural products for human therapeutics, where more than 50% are derived from natural products, only 11% of pesticides are derived from natural sources. Nevertheless, natural product pesticides have a potential to play an important role in controlling pests in both conventional and organic farms. Secondary metabolites produced by microbes (bacteria, actinomycetes and fungi) provide novel chemical compounds which can be used either alone or in combination with known compounds to effectively control insect pests and to reduce the risk for resistance development. There are several well-known examples of microbial natural products that are successful as agricultural insecticides (Thompson et al., 2000; Arena et al., 1995; Krieg et al. 1983).

The development of a microbial pesticide starts with the isolation of a microbe in a pure culture. It then proceeds with efficacy and spectrum screening using in vitro, in vivo or pilot scale trials in a greenhouse and in the field. At the same time, active compounds produced by the microbe are isolated and identified. For the commercialization of a microbial pesticide, the microbe has to be economically produced by fermentation at an industrial scale and formulated with biocompatible and approved additives to increase efficacy and to maximize the ease of application as well as storage stability under field conditions.

Chromobacterium

In 2000, Dr. Martin and her coworkers at USDA isolated a purple-pigmented bacteria (PRAA-4-1) from a forest soil in Maryland (Martin et al., 2007a). In the initial screening, they found this bacteria to be toxic to Colorado potato beetle and other insect pests (Martin et al., 2007b). This motile, Gram-negative, bacteria was identified as a new species of Chromobacteria, Chromobacterium substsugae sp. nov (Martin et al., 2007c). It is a facultatively aerobic, motile, Gram-negative beraproteobacterium with polar flagella. Colonies formed at 2-3 days on an L-agar plate at 25° C. are initially cream colored, gradually turning light to dark violet during the following 24 hours. Colonies of PRAA4-1 grow well on peptone based media with an optimum at 25° C., pH 6.5-8.0. and with 0-15% (w/v) NaCl (Martin et al., 2007a).

Since the finding of C. substugae by Martin and her coworkers, at least three new species of Chromobacteria have been isolated, and characterized; Young et al. (2008) isolated a novel Chromobacterium species, C. aquaticum, from spring water samples in Taiwan, and Kampfcr et al. (2009) isolated two species, C. piscinae and C. pseudoviolaceum, from environmental samples collected in Malaysia.

Of all known species of Chromobacteria, C. violaceum, a gram-negative saprphyte from soil and water. Published information on secondary metabolites produced by Chromobacteria is based on studies on C. violaceum only (see, for example, Durán and Menck (2001) for a comprehensive review of the pharmacological and industrial perspectives of C. violaceum). It is normally considered nonpathogenic to humans, but AS an opportunistic pathogen, it has occasionally been the causative agent for septicemia and fatal infections in humans and animals. C. violaceum is known to produce a purple pigment, violacein, which is a bisindole molecule generated by a fusion two L-tryptophan molecules in the presence of oxygen (Hoshino et al., 1987; Ryan and Drennan; 2009), Violacein biosynthesis is regulated by quorum-sensing, a common mechanism regulating various other secondary metabolism pathways in Gram-negative bacteria (McClean et al., 1997).

Other known metabolites of C. violaceum summarized by Durán and Menck (2001) include hydrogen cyanide, ferrioxamine E, B-lactamic glycopeptides SQ28,504 and SQ28,546, antibiotics such as aerocyanidin, aerocavin, 3,6-dihydroxy indoxazene, and monobactam SB-26,180, and an antitumoral depsipeptide FR901228. According to the review article by Durán and Menck (2001), C. violaceum also produces unusual sugar compounds such as extracellular polysaccharides and lipopolysaccharides.

US patent application publication no. US20120100236 also discloses compounds obtainable or derived from Chromobacterium species, more particularly, Chromobacterium substugae.

Mites and Acaricides

Tetranychus urticae (Two spotted spider mite) is a member of the Tetranychidae family. Spider mites are perhaps the most important mite pests of ornamentals. They also cause considerable damage in more than 180 species of greenhouse and field crops. These mites are also among the most difficult arthropod pests to control and resistance to chemicals can develop quickly (Stamps and Osborne 2009, Osborne, Ehler and Nechols, 1999).

Acaricides are compounds that kill mites (miticides) and ticks (ixodicides). This class of pesticides is large and includes antibiotics, carbamates, formamidine acaricides, pyrethroids, mite growth regulators, and organophosphate acaricides. Besides chemical pesticides, diatomaceous earth and fatty acids can be used to control mites. They typically work through disruption of the cuticle, which dries out the mite. In addition, some essential oils such as peppermint oil are used to control mites. In spite of the great variety of known acaricide compounds, mites remain a serious problem in agriculture because of the damage they cause to the crops. They can produce several generations during one season, which facilitates rapid development of resistance to the acaricide products used. Hence, new pesticide products with new target sites and novel modes of action are critically needed.

House Files

Musca domestica (House flies) are members of the family Muscidae. This family is considered an economic problem domestically and worldwide. Other members of the Muscidae family include face fly, stable fly, and horn fly. They are considered a nuisance and are vectors of human and animal diseases. Their habits of walking and feeding on garbage and excrement and on the humans and food make them ideal agents for the transfer of disease organisms. This species can also be a pest to animals and transmit disease through open wounds.

Plant Feeding Flies—Spotted Wing Drosophila

The spotted wing Drosophila, Drosophila suzakii is a recent invader to the fruit and vegetable growing areas in the United States. It is far more destructive than a well known related species Drosophila melanogaster and other Drosophila because D. suzukii can feed on and damage in-tact fruits and vegetables, while other Drosophila only feed on decaying plant material.

Root Maggots

Root maggots of the family Anthomyidae feed on the roots of several different plants. Cabbage Root Maggots affect cabbage, cauliflower, broccoli, and Brussels sprouts. (This group of vegetables is also known as ‘cole crops’). Different types of root maggots also occur that affect carrots, unions, and other vegetable crops. Because cole crops are cool-season vegetables, Cabbage Root Maggots are much more prominent in Northern zones of the US. They are difficult to control, because they hatch and feed underneath the soil, so you may only know they are there when you notice stunted growth or wilting foliage.

Green Peach Aphids

Myzus persicae, (green peach aphids) are members of the Aphididae family (see US20110054022). As evident by its common name, green peach aphids are pests of a wide range of fruits, vegetables and ornamental plants and have a worldwide presence. These insects are particularly harmful since they not only cause direct damage by feeding on phloem sap but are also potential vectors for the plum pox virus, the causal agent for Sharka disease, which causes fruit deformation and discoloration. As a result, infected trees must be uprooted. Attempts have been made to control these pests with various pesticides. However, resistance is often developed.

Potato Psyllid

Bactericera cockerelli, (potato psyllid) is a member of the Triozidae family and is a causative agent of zebra chip disease via infestation of gram negative bacteria. Although it is native to North America, it has been found in New Zealand as well (www.biosecurity.govt.nz/files/pests/potato-tomato-psyllid/psyillid-factsheet.pdf). The potato psyllid generally breeds in solanaceous hosts (such as tomatoes and potatoes). However, they have been found in other plants as well such as capsicum, chilli, eggplant, kumara, porporo, tamarillo and thornapple.

Litter Beetles

Alphitobins diaperinus is a serious pest in the poultry industry and is a member of the Tenebrionidae family. The Bt strain PS86B1 reportedly has activity against Alphitobius (U.S. Pat. No. 5,100,665 to Hickle et al.). Bt tenebrionis may have activity against larvae of this beetle as well (U.S. Pat. No. 5,244,660). Litter beetles and a few other coleopteran species act as vectors for protozoan, bacterial, and vital diseases of chickens and turkeys resulting in significant economic loss. Litter beetles act as a significant reservoir for pathogenic Salmonella species including the more pathogenic varieties, such as S. enterica serotype enteritidis. The problem is that poultry contaminated with pathogenic organisms like Salmonella threaten human health. These beetles inhabit the litter, wood, Styrofoam, fiberglass, and polystyrene insulation panels of chicken houses. Larvae and adult beetles thrive both on bird droppings and on grains used as chicken feed. These large beetle populations and their diverse habitats within chicken houses make it more difficult to eradicate the Salmonella they carry. In the midst of a heavy litter beetle infestation, or prior to establishing new chicken populations neither frequent changes in the litter nor dusting with multiple chemical insecticides is a completely effective control for this pest.

Grubs and Scarabs

Grubs, such as white grubs (Cyclocephala lurida), Southern Masked Chafer. (Rhizotrogus majalis) Japanese beetle larvae, (Popillia japonica) black vine weevil larvae (Otiorhynchus sulcatus),oriental beetle larvae (Anomala orientalis), members of the Scarabaeidae family, have been found to infest turf and pasture grasses. Adult scarabs have been found to infest ornamental plants, and numerous crops around the world. Various pesticides have been tried and include chemical pesticides, nematodes (see, for example, U.S. Pat. No. 7,641,573) and Bacillus thuringiensis (see U.S. Pat. No. 5,185,158), peromones, and natural repellents such as catnip and chives.

Polyhydroxyalkanoates (PHAs)

Bio-plastic is defined as a form of plastic synthesized from renewable resources such as plant starch and microbial species. Some of the biodegradable plastic materials under development include polyhydroxyalkanote (PHA), polylactide, aliphatic polyesters, polysaccharides, and the copolymers and/or blends of these. PHAs in particular include several polymeric esters such polyhydroxybutyrates, polyhydroxybutyrate co-hydroxyvalerates (PHBV), polyhydroxybutyrate co-hydroxyhexanoate (PHBHx) and polyhydroxybutyrate co-hydroxyoctonoate (PHBO). Poly3-hydroxybutyric acid (PHB) is the most common natural microbial PHA. Polyhydroxyalkanoates are 100% biodegradable polymers. Since they have similar properties to various synthetic thermoplastic like polypropylene, PHAs can be used in their place. They are also totally degraded to water and carbon dioxide under aerobic conditions and to methane under anaerobic conditions by micro-organisms in soil, lake water, sewages and sea water. Depending on the number of carbon atom in the chain. PHAs have been divided into two groups; short-chain length (SCL) which consists of 3-5 carbon atoms, and medium-chain length (MCL) which consists of 6-14 carbon atoms (Khanna S. Srivastava AK. 2005). These differences are mainly due to the substrate specificity of the PHA synthases that can accept 3HAs of a certain range of carbon length. The other well-known PHA_(SCL) is the copolymer, poly (3hydroxybutyrate-co-3-hydroxyvalerate) P(3HB-co-3HV), which comprise of four- and five-carbon monomeric units. The proportion of these monomeric units can vary, and this affects the physical properties of the polymer, i.e. less brittle with increasing proportion of 3HV unit.

In some microbial species, accumulation of PHA occurs during the presence of excess carbon and a limitation of nitrogen sources (Verlinden et al., 2007). PHAs produced in response to stressful conditions serve as energy storage molecules to be utilized when common energy sources are absent (Solaiman and Ashby, 2005). The plastic polymers accumulate intracellularly as light retracting amorphous storage granules in these organisms (Mukhopadhyay et al., 2005). PHB is synthesized from acetyl-CoA using three enzymatic steps (Krans et al., 1997). From a biotechnological point of view, the ability of bioplastics to be biodegradable makes them a desirable substitute for petrochemical-based plastic, an environmental pollutant (Lee, 1996). Increased production of bioplastics can significantly reduce carbon dioxide emissions, curtail plastic waste generation and decrease consumption of fossil fuels.

PHAs can be obtained from the following three methods: biosynthesis by microorganisms, photosynthesis by transgenic plants, and in vitro biosynthesis using appropriate enzymes (see, for example, U.S. Pat. No. 7,455,999, WO9914313). In most bacteria, cells synthesize PHA under growth-limiting substrates other than carbon source such as nitrogen, phosphorus or oxygen.

Accumulated PHA serves as both carbon and energy source during starvation. PHA also serves as a sink for reducing power and could therefore be regarded as a redox regulator within the cell. PHAs are also useful as stereo regular compounds which can serve as chiral precursors for the chemical synthetic of optically active compounds. Such compoumds are particularly used as biodegradable carriers for long-term dosage of drugs, medicines, hormones, insecticides and herbicides (Reddy 2003). They are also used as osteosynthetic materials in the stimulation of bone growth owing to their piezoelectric properties, in bone plates, surgical sutures and blood vessel replacements (Schaefer et al., 2000). Furthermore, there have been disclosures of method of copolymer production by microbiological process using various bacteria e.g. Alcaligenes eutrophus NC1MB 40124 (EP.0431883A2) and U.S. Pat. No. 7,455,999. EP No. 2236089A1 discloses uses of these polymers in multizone implants for orthopedic repair devices and soft tissue fixation devices. WO 91/00917A1 discloses method for controlling and modifying novel polyester biopolymer by manipulation of the genetics and enzymology of synthesis of polyhydroxybutyrate (PHB) and polyhydroxyalkanoate (PHA) polyesters at the molecular level in prokaryotic and eukaryotic cells, especially plants. WO 2005/030482A 1 discloses methods and uses as compostable packing materials. WO2008/110541 discloses the method of stabilization of polyhydroxybutyrates against thermal degradation.

Lignin

Lignin is a principal constituent of the woody structure of higher plants. Processed lignin is obtained as a by-product of wood pulping reactions. Lignin products include, for example, lignin sulphonates, alkali lignins, and oxylignins which may be obtained from sulphite, sulphate, and alkali waste liquors (Snook, 1982, Handbook for Pulp & Paper Technologists, TAPPI, Atlanta).

Lignin has been found to have a variety of commercial uses. For example, alkali soluble lignin has been used as a dispersing agent. U.S. Pat. No. 3,726,850 discloses the use of an alkali soluble, ozone-treated lignin product, which is essentially free of organically bound sulfur, as a dispersing agent for clays, dyestuffs, pesticides, carbon black and other materials. U.S. Pat. No. 4,666,522 discloses the use of lignosulphonate products for preparing emulsions of waxes, oils, fats, asphalts, and mixtures thereof. Lignin acetate, has been reported to be useful for applications such as acting as a binder in water-based printing ink compositions. (See, e.g., U.S. Pat. No. 4,612,051). U.S. Pat. No. 5,668,183 discloses the use of lignin sulphonate products for dispersing fat-soluble substances. Furthermore, there have been disclosures of binding of lignin-pesticide complexes (see, for example, U.S. Pat. No. 3,813,236, U.S. Pat. No. 3,929,453, reissued as Re. No. 29,238, U.S. Pat. No. 4,381,194, US Patent Application Pub. No. 20110015237, US Patent Application Pub. No. 2010136132, US Patent Application Pub. No. 20100278890, US Patent Application Pub. No. 20080113920. US Patent Application Pub. No. 2006247130. U.S. Pat. No. 7,867,507, WO2003/005816, U.S. Pat. No. 5,994,266).

Sodium Benzoate

Sodium benzoate has been used in various formulations as an anti-microbial in food preparations. For example, U.S. Pat. No. 6,599,514 discloses synergistic antifungal compositions comprising an antifungal agent and a food additive, which produces a synergistic effect on the overall antifungal activity or the antifungal composition. Food additives disclosed in U.S. Pat. No. 6,599,514 included sorbic acid and sorbates, benzoic acid and benzoates, hydroxy-benzoates, sulphur dioxide and sulphites, biphenyl and derivatives, nitrites, nitrates, lactic acid, lactates, citric acid and citrates, tartaric acid and tartrates, orthophosphoric acid and orthophosphates, malates, adipic acid, succinic acid, 1,4-heptonolactone, nicotinic acid, triammoniun citrate, ammonium ferric citrate, calcium disodium EDTA, glycerol, di-, tri- and polyphosphates, fatty acids (E470), mono- and diglycerides of fatty acids (E471), esters of mono- and diglycerides of fatty acids, carbonates, gluconates, chlorine (E92S), sodium hexametaphosphate, butylated hydroxyanisole (BHA), butylated hydroxy toluene (BHT)(E321), t-butyl hydroquinone (THBQ), propyl gallate, calcium heptonate, calcium phytate, diethyl ether, EDTA, disodium dihydrogen EDTA, ethyl acetate, glycerol mono-, di- and triacetates, glycine, oxystearin, propan-1,2-diol and propan-2-01 and sodium heptonate.

Sodium benzoate has also been used in pesticide formulations. For example, U.S. Pat. No. 4,668,507 by S C Johnson teaches use of sodium benzoate in pesticides contained in pressurized steel aerosol delivery systems where main mode of stabilization is corrosion inhibition. U.S. Pat. No. 5,620,678 discloses an insecticide formulation that include sodium benzoate as corrosion inhibitor. U.S. Pat. No. 4,731,379 teaches insecticidal compositions that contain sodium benzoate when used as an animal shampoo to kill fleas. In this patent the use of sodium benzoate is not shown to increase effectiveness of the insecticide or stabilize the product but rather, is thought to assist in healing of wounds of the treated animal. U.S. Pat. No. 5,017,620 teaches insecticidal compositions that contain sodium benzoate and other known preservatives when used as an anti-microbial to stabilize the product while in storage. U.S. Pat. No. 6,841,572 discloses an aqueous solution for treating live plants, crops, trees, pre-harvest fruits, vegetables, leaves, stems, roots and flowers having it pH of between 4.0 and 6.5 and consisting essentially of fungicidally and/or bactericidally effective concentrations of one or more preservative compounds selected from the group consisting of sorbic, benzoic and lactic acid; the sodium, potassium, calcium and ammonium salts of benzoic, sorbic, hydroxymethyl glycinic, lactic and propionic acid; and methyl, ethyl, propyl and buryl paraben, at least one anionic surfactant, and optionally an acidulant.

SUMMARY

Provided are compositions and methods for modulating and/or infestation of one or more Acari (arachnid), Muscidae, Drosophilidae, Anthomyidae, Aphididae, Triozidae. Tenebrionidae and/or Scarabaiedae pests comprising or using a supernatant, filtrate and/or extract and/or one or more metabolites from said supernatant, filtrate and/or extract of a strain of Chromobacterium sp., particularly a violacein producing strain, including but not limited to Chromobacterium piscinae, C. pseudoviolaceum, Chromobacterium substugae and more particularly, a strain of Chromobacterium substugae sp. nov and even more particularly a strain of Chromobacterium substugae sp. nov. having the identifying characteristics of NRRL B-30655 described in U.S. Pat. No. 7,244,607.

Specifically provided is a method for modulating infestation of arachnid (Acari or Acarina) and/or one or more insect pests belonging to the Anthomyidae, Drosophilidae, Muscidae, Aphididae, Triozidae, Tenebrionidae or Scarabaiedae family in a location where modulation is desired an amount of (a) a supernatant, filtrate and/or extract and/or one in more metabolites from said supernatant, filtrate and/or extract of a strain of Chromobacterium sp. and (b) another pesticidal substance, particularly, an acaracide and/or insecticide which may be effective against Acari and/or one or more insect pests belonging to the Anthomyidae, Drosophilidae, Muscidae, Aphididae. Triozidae, Tenebrionidae or Scarabaiedae family effective to modulate infestation of arachnid and/or one or more insect pests belonging to the Anthomyidae, Drosophilidae, Muscidae, Aphididae, Triozidae, Tenebrionidae or Scarabaiedae family at said location.

In a specific embodiment, the Acari infestation is Tetranychidae (mite) infestation. In a mote specific embodiment, the mite infestation is Tetranychus urticae infestation.

In another specific embodiment, the insect pest infestation is a Musca sp., Myzus sp., Bactericera sp. Cyclocephala sp or Alphitobius sp. Drosophila sp. Delia sp. Rhizotrogus sp. Popillia sp. Anomala sp. or Otiorhynchus sp. infestation. In an even more specific embodiment, the insect pest infestation is a Musca domesitcas (house fly), Drosophila suzukii (Spotted Wing Drosophila), Delia radicum (cabbage root maggot), Myzus persicae, (green peach aphid), Bactericera cockerelli (potato psyllid), Alphitobius diaperinusxi (litter beetle), Cyclocephala lurida (white grub), Rhizotrogus majalis (Southern Masked Chafer), Popilla japonica (Japanese beetle), Otiorhynchus sulcatus (black vine weevil), Anomala orientalis (oriental beetle).

Also provided herein is a pesticidal combination which modulates infestation of at least one arachnid and/or one or more insect pests belonging to the Acari, Anthomyidae, Drosophilidae, Muscidae, Aphididae, Triozidae, Tenebrionidae or Scarabaiedae family comprising as active components: (a) a supernatant, filtrate and/or extract of Chromobacterium sp. and/or one or more metabolite(s) from said supernatant, filtrate and/or extract of Chromobacterium sp. and (b) anther pesticidal substance, particularly, an acaracide and/or insecticide which may be effective against one or more insect pests belonging to the Acari, Anthomyidae, Drosophilidae, Muscidae, Aphididae, Triozidae, Tenebrionidae or Scarabaiedae family wherein (a) and (b) may optionally be present in synergistic amounts. The pesticidal substance may be (a) derived from a microorganism: (b) a natural product and/or (c) a chemical pesticide and in particular a chemical insecticide.

In one embodiment, the metabolite may be a compound that (a) has pesticidal activity, (b) has a molecular weight of about 840-900 as determined by Liquid Chromatographv/Mass Spectroscopy (LC/MS) and (c) has an High Pressure Liquid Chromatography (HPLC) retention time of about 7-12 minutes on a reversed phase C-18 HPLC column using a water:acetonitrile (CH₃CN) gradient solvent system (0-20 min; 90-0% aqueous CH₃CN, 20-24 min; 100% CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90% aqueous CH₃CN) at 0.5 mL/min flow rate and UV detection of 210 nm and (d) is optionally obtainable from a Chromobacterium species. The compound in one embodiment may be a peptide.

In a particular embodiment, the compound has 43 cartons, seven methyl, ten methylene carbons, twelve methines, 6 olefinic methines, and eight quaternary carbons determined by ¹³C NMR. In more particular embodiments. the compound encompasses compounds “A”, “B”, “C”, “D”, depicted as ##STR001#, ##STR001a##, ##STR001b##, ##STR001c## respectively.

In one specific embodiment, the compound “A”: (a) is obtainable from a Chromobacterium species; (b) is toxic to a pest; (c) has a molecular weight of about 840-890 and more particularly, 860 as determiner) by liquid Chromatography/Mass Spectroscopy (LC/MS): (d) has ¹H NMR values of δ 8.89, 8.44, 8.24, 8.23, 7.96, 7.63, 6.66, 5.42, 5.36, 5.31, 5.10, 4.13, 4.07, 4.05, 3.96, 3.95, 3.88, 3.77, 3.73, 3.51, 3.44, 3.17, 2.40, 2.27, 2.11, 2.08, 2.03, 2.01, 1.97, 1.95, 1.90, 1.81, 1.68, 1.63, 1.57, 1.53, 1.48, 1.43, 1.35, 1.24, 1.07, 1.02, 0.96, 0.89, 0.88, 0.87, 0.80 and has ¹³C NMR values of δ 173.62, 172.92, 172.25, 172.17, 171.66, 171.28, 170.45, 132.13, 130.04, 129.98, 129.69, 129.69, 125.48, 98.05, 70.11, 69.75, 68.30, 68.25, 64.34, 60.94, 54.54, 52.82, 49.72, 48.57, 45.68, 40.38, 39.90, 38.18, 36.60, 31.98, 31.62, 31.58, 29.53, 28.83, 27.78, 24.41, 23.06, 22.09, 20.56, 19.31, 18.78, 17.66, 15.80 (e) has an High Pressure Liquid Chromatography (HPLC) retention time of about 7-12 minutes, more specifically about 9 minutes and even more specifically about 9.08 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5μ C18(2) 100 A, 100×4.60 mm) column using a water:acetonitrile (CH₃CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH₁₃CN, 20-24 min; 100% CH₁₃CN, 24-27 min; 0-90% aqueous CH₁₃CN, 27-30 min, 90% aqueous CH₁₃CN) at 0.5 mL/min flow rate and UV detection of 210 nm.

In another specific embodiment, the compound “B” has the following characteristics: (a) is obtainable from a Chromobacterium species; (b) is toxic to a pest; (c) has a molecular weight of about 850-900 and more particularly, 874 as determined try Liquid Chromatography/Mass Spectroscopy (LC/MS): (d) has an High Pressure Liquid Chromatography (HPLC) retention time of about 7-12 minutes, more specifically about 9 minutes and even more specifically about 954 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5μ C18(2) 100 A, 100×4.60 mm) column using a water:acetonitrile (CH₃CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH₁₃CN, 20-24 min; 100% CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-90% min; 90% aqueous CH₃CN) at 0.5 mL/min flow rate and UV detection of 210 nm.

The metabolite may also be a compound including but not limited to:

(A) a compound having the structure ##STR001##

or a pesticidally acceptable salt or stereoisomers thereof, wherein R is —H, lower chain alkyl containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl moieties, aryl or arylalkyl moiety, substituted lower alkyl: X is O, NB, NR or S; n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ are each independently H, are the same or different and independently an amino acid side-chain moiety or an amino acid side-chain derivative, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl;

(B) a compound having the structure ##STR001a##

wherein R is —H, lower chain alkyl containing 1, 2, 3, 4, 5, 6, 8 or 9 alkyl moieties, aryl or arylalkyl moiety, substituted lower alkyl; X is O, NH, NR or S; R_(2a), R_(2b) are independently selected from the group consisting of —H, alkyl, lower-alkyl, substituted alkyl and substituted lower-alkyl; R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ are each independently H, are the same or different and independently an amino acid side chain moiety or an amino acid side-chain derivative, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioelkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.

(C) a compound having the structure ##STR001b##

wherein R —H, lower chain alkyl containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl moieties, aryl or aryl alkyl moiety, substituted lower alkyl; X is O, NH, NR or S: n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R_(2a), R_(2b) are independently selected from the group consisting of —H, alkyl, lower-alkyl, substituted alkyl and substituted lower-alkyl; R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ are each independently H, are the same or different and independently an amino acid side-chain moiety or an amino acid side-chain derivative, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.

(D) a compound having the structure ##STR001c##

wherein R is —H, lower chain alkyl, aryl or aryl alkyl moiety, substituted lower alkyl containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl moieties; X is O, NH, NR or S; n is 0, 1, 2, 3, 4, 5, 6, 7, 8 or 9; R2a, R2b are independently selected from the group consisting of —H, alkyl, lower-alkyl, substituted alkyl and substituted lower-alkyl; R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R₁₀, R₁₁ are each independently H, are the same or different and independently an amino acid side-chain moiety or an amino acid side-chain derivative, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.

In a more particular embodiment, the metabolite is chromamide A (1).

In a particular embodiment, the metabolite is compound “C”, has the following characteristics: (a) is obtainable from a Chromobacterium species; (b) is toxic to one or more pests; (c) has a molecular weight of about 325-360 and more particularly, about 343 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (d) has an High Pressure Liquid Chromatography (HPLC) retention time of about 8-14 minutes, more specifically about 10 minutes and even more specifically about 10.88 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5μ C18(2) 100 A, 100×4.60 mm) column using a water:acetonitrile (CH₃CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH₃CN, 20-24 min; 100% CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90%- aqueous CH₃CN) at 0.5 mL/min flow rate and UV detection of 210 nm. In a particular embodiment, compound “C” may be violucein (2), a known compound isolated earlier from Chromobacterium violaceum.

In another embodiment, another metabolite used in the compositions and methods set forth above, is the compound “D”, has the following characteristics: (a) is trainable from a Chromobacterium species; (b) is toxic to a pest; (c) has a molecular weight of about 315-350 and more particularly, about 327 as determined by Liquid Chromatography/Mass Spectroscopy (LC/MS); (d) has an High Pressure Liquid Chromatography (HPLC) retention time of about 10-15 minutes, more specifically about 12 minutes and even more specifically about 12.69 min on a reversed phase C-18 HPLC (Phenomenex, Luna 5μ C18(2) 100 A. 100×4.60 mm) column using a water:acetonitrile (CH₃CN) with a gradient solvent system (0-20 min; 90-0% aqueous CH₃CN, 20-24 min; 100% CH₃CN, 24-27 min; 0-90% aqueous CH₃CN, 27-30 min; 90% aqueous CH₃CN) at 0.5 mL/min flow rate and UV detection of 210 nm. In a particular embodiment, compound “D” may be characterized as deoxyviolacein (3), a known compound isolated earlier from Chromobacterium violaceum.

In another specific embodiment, the compound may have the following structure

wherein R is —H, lower chain alkyl containing 1, 2, 3, 4, 5, 6, 7, 8 or 9 alkyl moieties, aryl or aryl alkyl moiety, substituted lower alkyl, halogens; R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉ are each independently H, are the same or different, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy, substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.

Further provided is a method for (1) modulating pest (e.g., nematode, insect, soil-borne bacteria) infestation in a plant comprising applying to the plant and/or seeds thereof and/or substrate used for growing said plant or a method for modulating soil borne bacteria in soil comprising applying to the plant, seeds, and/or substrate an amount of (1) a compound that

(a) has pesticidal and/or antimicrobial activity;

(b) has a molecular weight of about 950-1450 as determined by LTQ Orbitrap XL hybrid Fourier Transform Mass Spectrometer.

(c) has ¹H NMR δ values of 5.22 (sext, 1H, 2.62 (dd, 1H), 2.53 (dd, 1H),and 1.31 (d, 3H) (d) has ¹³C NMR δ values of 169.2, 67.6, 40.9, and 19.8.

(d) comprises the structure-(—O—CHCH₃-CH₂-CO—)_(n)-, where n=6-50

(c) is obtainable from a Chromobacterium species and

(II) optionally another pesticidal substance effective to modulate infestation in said plant. Compound (1) may have the structure:

Wherein

X, is independently O, —NR, or —S, wherein R is H or C₁-C₁₀alkyl; Y, is independently —O, —S; n=6-50; R₁, R₂ are each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, cycloalkyl, substituted cycloalkyl, alkoxy substituted alkoxy, thioalkyl, substituted thioalkyl, hydroxy, halogen, amino, amido, carboxyl, —C(O)H, acyl, oxyacyl, carbamate, sulfonyl, sulfonamide, or sulfuryl.

In particular, the compound (1) has the structure:

wherein n=10-25. In a most specific embodiment, (1) is an alpha- butyric acid.

Further provided is a method for obtaining the compounds set forth above. The method comprises culturing a strain of a Chromobacterium sp in a whole cell broth under conditions sufficient to produce the compound and dilating the compound produced from the whole cell broth.

In a related aspect, disclosed are methods for stabilizing biological pesticide compositions against physical separation and loss of activity due to exposure to sunlight by applying an amount of a stabilizing agent to said biological pesticide composition effective to stabilize the biological pesticide composition against physical separation and loss of activity due to exposure to sunlight Also provided are compositions comprising such agents. In a particular embodiment, the pesticidal composition comprises Chromobacterium sp. filtrate, supernatant, extract or pesticidally active substance derived therefrom which may be present in the amount of at least about 0.5% and particularly between about 0.3 wt % based on dry cell weight to about 30 wt. %. The stabilizing agent may in a particular embodiment be a benzoic acid salt and/or lignin salt, particularly lignin sulfonate salt, including but not limited to sodium, potassium, calcium, magnesium, ammonium, and combinations thereof and may present in the amount of at least about 2.5 wt % and may preferably be present in the amount of about 5%-15%.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of purification scheme for obtaining chromamide A (1) violacein (2) and deoxyviolacein (3), from culture broth.

FIG. 2 depicts chemical structures for chromamide A (1) violacein (2) and deoxyviolacein (3).

FIG. 3 shows results from MBI-203 TSSM Leaf disk assays. CFD represents Cells Freeze Dried, which were then reconstituted in water to represent a ½x, 1x and 2.5x dose of the original Whole Cell Broth.

FIG. 4 shows the mean number of eggs laid by potato psyllid females exposed to MBI-203 treated pepper leaf.

FIG. 5 shows the mean number of eggs oviposited by psyllid females 5 days post exposure to MBI-203 treated and untreated pepper leaf discs.

FIG. 6 shows the mean number of Green Peach Aphid (GPA) nymphs and adults on treated pepper leaf discs 24 hours post exposure of adults (P<0.0001, LSD, α=0.05). Means of the same letter not statistically different from each other, dH2O-negative control, 203 10% v/v-positive control.

FIG. 7 shows the mean number of Green Peach Aphid progeny (nymphs) on MBI-203 treated pepper leaf discs 3 days post exposure of adults (P<0.0001, LSD, α=0.05). Means of the same letter not statistically different from each other, dH₂O-negative control, Avid 10% v/v-positive control.

FIG. 8 shows the mean number of Green Peach Aphid progeny (nymphs) on MBI-203 and DF2 (MBI-203 formulated product) treated pepper leaf discs 3 days post exposure of adults (P<0.0001, LSD, α=0.05). Means of the same letter not statistically different from each other, dH₂O-negative control, Avid 10% v/v-positive control.

FIG. 9 is a schematic representation of purification scheme for violacein (2) and oligo (β-hydroxybutyric acid (4), from culture broth.

FIG. 10 depicts the results of nematocidal bioassays of various fractions and of compound oligo-hydroxybutyric acid (1).

FIGS. 11A and 11B show the mean number of green peach aphid, Myzus persicae (nymphs and adults) on treated pepper leaf discs 24 hours post exposure. Means of the same letter are not significantly different from each other (LSD, P<0.0001, α=0.05), 203 Me-MBI-203 crude extract in methanol solvent. Me-Methanol (blank treatment), 203, Ace-MBI-203 crude extract in acetone solvent, Ace-Acetone (blank treatment), dH₂O-negative control, Avid 10% -positive control.

FIGS. 12A and 12B show the mean number of green peach aphid, Myzus persicae (nymphs and adults) on treated pepper leaf discs 24 hours post exposure. Means of the same letter are not statistically different front each other. Ace-acetone only (blank treatment, DCM-, EA-, Wash-, MeOH-, dH₂O-negative control, 203 10% - MBI-203 std (positive control).

FIG. 13 shows the mean number of Green Peach Aphid progeny (nymphs) on treated pepper leaf discs 3 days post exposure of adults (P<0.0018, LSD, α=0.05). Means of the same letter not statistically different from each other. VL1-violacein at 0.5 μg/mL, VL2-violacein at 10 μg/mL, Acetone-blank treatment, dH₂O negative control, Avid 10% v/v-positive control.

FIG. 14 shows the mean number of Green Peach Aphid (GPA) nymphs and adults on treated pepper leaf discs 24 hours post exposure of adults (P<0.0001, LSD, α=0.05). Means of the same letter not statistically different from each other. dH₂O-negative control, 203 10% v/v-positive control.

FIG. 15 shows the mean number of Green Peach Aphid (CPA) nymphs and adults on treated pepper leaf discs 24 hours post exposure of adults (P<0.0001, LSD, α=005). Means of the same letter not statistically different from each other. dH₂O-negative control, 203 10%, v/v-positive control.

FIG. 16 shows MBI-203@3% presun vs postsun mortality, zeroed to water.

FIG. 17 shows MBI-203: Cabbage Aphid plant spray EP/EP+ NaBenz.

FIG. 18 shows MBI-203 EP+− Na Benz, Cabbage Aphids plant spray, corrected.

FIG. 19 shows MBI-203: EP+ CaCO3/NaBenz PRE/POST SUN, normalized to negative control.

FIG. 20 shows MBI-203 EP SDP post sun, 3 and 6 day assays.

FIG. 21 shows MBI-203 EP w/Na Benz+lignin in sun, assay day 4, corrected.

FIG. 22 shows MBI-203 EP w/Na Benz+lignin in sun, percent activity loss, assay day 4.

FIG. 23 shows MBI-203: EP+liguin+sun, Broccoli CL assay day 3.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

While the compositions and methods heretofore are susceptible to various modifications and alternative forms, exemplary embodiments will herein be described in detail. It should be understood, however, that there is no intent to limit the invention to the particular forms disclosed, but on the contrary, the intension is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention as defined by the appealed claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is included therein. Smaller ranges are also included. The upper and lower limits of these smaller ranges are also included therein, subject to any specifically excluded limit in the stated range.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

It must be noted that as used herein and in the appended claims, the singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.

As defined herein, “derived from” means directly isolated or obtained from a particular source or alternatively having identifying characteristics of a substance or organism isolated or obtained from a particular source.

A “carrier” as defined herein is an inert, organic or inorganic material, with which the active ingredient is mixed or formulated to facilitate its application to plant or other object to be treated, or its storage, transport and/or handling.

The term “modulate” as defined herein is used to mean to alter the amount of pest infestation or rate of spread of pest infestation.

The term “pest infestation” as defined herein, is the presence of a pest in an amount that causes a harmful effect including a disease or infection in a host population or emergence of an undesired weed in a growth system.

A “pesticide” as defined herein, is a substance derived from a biological product or chemical substance that increase mortality or inhibit the growth rate of plant pests and includes but is not limited to nematocides, insecticides, herbicides, plant fungicides, plant bactericides, and plant viricides.

A “biological pesticide” as defined herein is a microorganism with pesticidal properties.

Methods of Production

As noted above, the biological pesticide may comprise or tie derived from an organism having the identifying characteristics of a Chromobacterium species, more particularly, from an organism having the identifying characteristics of a strain of Chromobacterium substugae, more particularly horn a strain of Chromobacterium substugae sp. nov. which may have the identifying characteristics of NRRL B-30653, or alternatively from any other microorganism. The methods comprise cultivating these organisms and obtaining the compounds and/or compositions of the present invention by isolating these compounds from the culture of these organisms.

In particular, the organisms are cultivated in nutrient medium using methods known in the art. The organisms may be cultivated by shake flask cultivation, small scale or large scale fermentation (including but not limited to continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in suitable medium and under conditions allowing cell growth. The cultivation may take place in suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available may be available from commercial sources or prepared according to published compositions.

After cultivation, the compounds and/or compositions of the present invention may be extracted from the culture broth. The extract may be fractionated by chromatography.

Compositions

The substances set forth above used in the compositions and methods disclosed herein can be formulated in any manner. Non-limiting formulation examples include but are not limited to Emulsifiable concentrates (EC), Wettable powders (WP), soluble liquids (SL), Aerosols, Ultra-low volume concentrate solutions (ULV), Soluble powders (SP), Microencapsulation, Water dispersed Granules, Flowables (FL), Microcmulsions (ME), Nano-emulsions (NB), etc. In any formulation described herein, percent of the active ingredient is within a range of 0.01 % to 99.99%.

The compositions may be in the form of a liquid, gel or solid. Liquid compositions comprise pesticidal compounds derived from a Chromobacterium strain. e.g. a strain having the identifying characteristics of Chromobacterium substugae sp. Nov and more particularly, having the identifying characteristics of NRRL B-30655 (sec U.S. Pat. No. 7,244,607).

A solid composition can be prepared by suspending a solid carrier in a solution of pesticidal compounds and drying the suspension under mild conditions, such as evaporation at room temperature or vacuum evaporation at 65° C. or lower.

A composition may comprise gel-encapsulated compounds derived from the Chromobacterium strain. Such gel-encapsulated materials con be prepared by mixing a gel-forming agent (e.g., gelatin, cellulose, or lignin) with a culture or suspension of live or inactivated Chromobacterium, or a cell-free filtrate or cell fraction of a Chromobacterium culture or suspension, or a spray- or freeze dried culture, cell, or cell fraction or in a solution of pesticidal compounds used in the method of the invention; and inducing gel formation of the agent.

The composition may additionally comprise a surfactant to be used for the purpose of emulsification, dispersion, wetting, spreading, integration, disintegration control, stabilization of active ingredients, and improvement of fluidity or rust inhibition. In a particular embodiment, the surfactant is a non-phytotoxic non-ionic surfactant which preferably belongs to EPA Inerts List 4B. In another particular embodiment, the nonionic surfactant is polyoxyethylene (20) monolaurate. The concentration of surfactants may range between 0.1-35% of the total formulation, preferred range is 5-25%. The choice of dispersing and emulsifying agents, such as non-ion to, anionic, amphoteric and cationic dispersing and emulsifying agents, and the amount employed is determined by the nature of rise composition and the ability of the agent to facilitate the dispersion of the compositions of the present invention.

The composition as set forth above also comprises a stabilizing agent, which stabilizes a biological pesticide composition against physical separation and loss of activity due to exposure to sunlight. This stabilizing agent may be a benzoic acid salt or lignin sulfonate salt.

The composition set forth above may be combined with another microorganism and/or pesticide (e.g, nematocide, fungicide, insecticide, antibiotic or anti-microbial agent). The microorganism may include but is not limited to an agent derived from Bacillus sp., Pseudomonas sp., Brevabacillus sp., Lecanicillium sp., non-Ampelomyces sp., Pseudozyma sp., Streptomyces sp. Burkholderia sp, Trichoderma sp, Gliocladium sp. Alternatively, the agent may be a natural oil or oil-pioduct having fungicidal and/or insecticidal activity (e.g., paraffinic oil, tea tree oil, lemongrass oil, clove oil, cinnamon oil, citrus oil, rosemary oil). Furthermore, the pesticide may be a single site anti-fungal agent which may include but is not limited to benzimidazole, a demethylation inhibitor (DMI) (e.g., imidazole, piperazine, pyrimidine, triazole), morpholine, hydroxypyrimidine, anilinopyrimidine, phosphorothiolate, quinone outside inhibitor, quinoline, dicarboximide, carboximide, phenylamide, anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid, hydroxyanilide, antibiotic, polyoxin, acylamine, phthalimide, benzenoid (xylylalanine), a demethylation inhibitor selected from the group consisting of imidazole, piperazine, pyrimidine and triazole (e.g., bitertanol, myclobutanil, penconazole, propicoazole, triadimefon, bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole, tebuconazole, tetraconazole), myclobutanil, and a quinone outside inhibitor (e.g., strobilurin). The strobilurin may include but is not limited to azoxystrobin, kresoxim-methoyl or trifloxystrobin. In yet another particular embodiment, the anti-fungal agent is a quinone, e.g., quinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether). The anti-fungal agent may also be derived from a Reynoutria extract.

The fungicide can also be a multi-site non-inorganic, chemical fungicide selected from the group consisting of chloronitrile, quinoxaline, sulphamide, phosphonate, phosphite, dithiocarbamate, chloralkythios, phenylpyridin-amine, cyano-acetamide oxime.

The composition may as noted above, further comprise an insecticide. Tbe insecticide may include but is not limited to avermeetin, Br, neem oil, spinosuds, Burkholderdia sp. as set forth in US Patent Appln. Pub. No. 2011-0207604, entomopathogeaic fungi such a Beauveria bassiana and chemical insecticides including but not limited to organochlorine compounds, organophosphorous compounds, carbamates, pyrethroids, and neonicotinoids.

As noted above, the composition may further comprise a nematocide. This nematocide may include but is not limited to avermeetin, microbial products such as Biome (Bacillus firmus). Pasteuria spp and organic products such as saponins.

Uses

The compositions, cultures and supernatants and pesticidal compounds set forth above may be used as pesticides. In particular, the compounds or compositions as set forth above may be used as insecticides, bactericides (against soil-borne bacteria) and nematocides. Specifically, nematodes that may be controlled using the method set forth above include but are not limited to parasitic nematodes such ax root-knot, cyst, and lesion nematodes, including but not limited to Meloidogyne sp. Tylenchorhynchus sp. Hoplolaimus sp. Helicotylenchus sp., Pratylenchus sp., Heterodera sp., Globodera, sp., Trichodorus sp. Paratrichodorus sp., Xiphinema sp., and Criconema sp.; particularly Meloidogyne incognita (root knot nematodes), as well as Globodera rostochiensis and Globodera pailida (potato cyst nematodes); Heterodera glycines (soybean cyst nematode); Heterodera schachtii (beet cyst nematode); and Heterodera avenae (cereal cyst nematode).

As noted above, the active ingredient(s) and compositions set forth above may also be applied to locations containing Acari (arachnids), such as mites, including but not limited to, Panonychus sp. such as Panonychus citri (citrus red mite), and Panonychus ulmi (red spider mite), Tetranychus sp. such as Tetranychus kanzawi (Kanzawa spider mite), Tetranychus urticae (2 spotted spider mite). Tetranychus pacificus (Pacific spider mite), Tetranychus turkestanii (Strawberry mite) and Tetranychus cinnabarinus (Carmine spider mite), Oligonychus sp. such as Oligonychus panicae (avacado brown mite), Oligonychus perseae (persea mite), Oligonychus pratensis (Banks grass mite) and Oligonychus coffeae, Aculus sp. such as Aculus cornatus (Peach silver mite), Aculus fockeni (plum rust mite) and Aculus lycopersici (tomato russet mite), Eotetranychus sp. such as Eotetranychus wilametri, Eotetranychus yumensis (yuma spider mite) and Eotetranychus sexmaculatis (6-spotted mite), Bryobia rubrioculus (brown mite), Epitrimerus pyri (pear rust mite), Phytoptus pyri (Pear leaf blister mite). Acalitis essigi (red berry mite), Polyphagotarsonemus lotus (Broad mite), Eriophyes sheldoni (citrus bud mite), Brevipulpus lewisi (citrus flat mite), Phylocopiruia oleivora (citrus rust mite), Petrobia lateens (Brown wheat mite), Oxyenus maxwelli (olive mite), Rhizoglyphus spp., Tyrophagus spp., Dipracus gigantorhyncus (bigheaded plum mite) and Penthaleoc major (winter grain mite), Avocado red mite, Flat mite, black and red Mango spider mite. Papaya leaf edgeroller mite, Texas citrus mite, European red mite, Grape crincum mite (blister mite). Pacific spider mite, Willamette spider mite; Pink citrus rust mite. Such locutions may include but are not limited to crops that are infested with such mites or other arachnids (e.g., aphenids). Such locations may include but are not limited to crops that are infested with such mites or other arachnids (e.g., aphenids).

Phytopathogenic insects controlled by the method set forth above include but are not limited to non-Culicidae larvae insects from the order (a) Lepidoptera, for example, Acleris spp., Adoxophyes spp., Aegeria spp., Agrotis spp., Alabama argillaceae, Amylois spp., Anticarsia gemmatalix.Archips spp., Argyrotaenia spp., Aufographa spp., Busseola fusca, Cadra cautella, Carposina nipponensis, Chilo spp., Choristoneura spp., Clysia ambiguella, Cnaphalocrocis spp., Cnephasia spp., Cochylis spp., Coleophora spp., Crocidolomia binotalis, Cryptophlebia leucotreta, Cydia spp., Diatraea spp., Diparopsis castanea, Farias spp., Ephestia spp., Eucosma spp., Eupoecilia ambiguella, Euproctis spp., Euxoa spp., Grapholita spp., Hedya nubiferana, Heliothis spp., Hellula undalis, Hyphantria cunea, Keiferia lycopersicella, Leucoptera scitella, Lithocollethis spp., Lobesia botrana, Lymantria spp., Lyonetia spp., Malacosoma spp., Mamestra brassicae, Manduca sexta, Operophtera spp., Ostrinia nubilalis, Pammene spp., Pandemia spp., Panolis flammea, Pectinophora gossypiella, Phthorimaea operculella, Pieris rapae, Pieris spp., Plutella xylostella, Prays spp., Scirpophaga spp., Sesamia spp., Sparganothis spp., Spodoptera spp., Synanthedon spp., Thaumetopoea spp., Tortrix spp., Trichoplusia ni and Yponomeuta spp.: (b) Coleoptera, for example, Agriotes spp., Alphitohius sp., Anomola spp. e.g., Anomala orientalis; Anthonomus spp., Atomaria linearis, Chaetocnema tibialis, Cosmopolites spp., Curculio spp., Cyclocephala spp., e.g., Cyclocephala lurida, Dermestes spp., Diabrotica spp., Epilachna spp., Eremnus spp., Leptinotarsa decemlineata, Lissorhoptrus spp., Melolontha spp., Orycaephilus spp., Otiorhynchus spp., Otiorhynchus suicatus, Phlyctious spp., Popillia spp., e.g., Popilla japonica, Psylliodes spp., Rhizopertha spp-, eg., Rhizotrogus majalis, Sitophilus spp., Sitotroga spp., Tenebrio spp., Tribolium spp. and Trogoderma spp.: (c) Orthoptera, for example, Blatta spp., Blattella spp., Gryllotalpa spp., Leucophaea maderae, Locusta spp., Periplaneta spp. and Schistocerca spp.: (d) lsoptera, for example, Reticulitermes spp., (e) Psocoptera, for example, Liposcelis spp.: (f) Anoplura, for example, Haematopinus spp., Linognathus spp., Pediculus spp., Pemphigus spp. and Phylloxera spp.; (g) Mallophaga, for example, Damalinea spp. and Trichodectes spp.: (h) Thysanoptera, for example, Frankliniella spp., Hercinotnrips spp., Taeniothrips spp., Thrips palmi, Thrips tabaci and Scirtothrips aurantii; (i) Heteroptera, for example, Cimex spp., Distantiella theobroma, Dysdercus spp., Euchistus spp., Eurygaster spp., Leptocorisa spp., Nezara spp., Piesma spp., Rhodnius spp., Sahlbergella singularis, Scotinophara spp. and Tniatoma spp.: (j) Homoptera, for example, Aleurothrixus floccosus, Aleyrodes brassicae, Aonidiella spp., Aphididae, Aphis spp., Aspidiotus spp., Bactericera spp., Bemisia tabaci, Ceroplaster spp., Chrysomphalus aonidium, Chrysomphalus dictyospermi, Coccus hesperidum, Empoasca spp., Eriosoma larigerum, Erythroneura spp., Gascardia spp., Laodelphas spp., Lecanlum corni, Lepidosaphes spp., Macrosiphus spp., Myzus spp., Nephotettix spp., Nilaparvata spp., Paratoria spp., Pemphigus spp., Planococais spp., Pseudaulacaspis spp., Pseudococcus spp., Psylla spp., Pulvinaria aethiopica, Quadraspidiotus spp. Rhopalosiphum spp., Saissetia, spp., Scapholdeus spp., Schizaphis spp., Sitobion spp., Trialeurodes vaporariorum, Triozidae spp., Trioza erytreae and Unaspis citri; (k) Hymenoptera, for example, Acromyrmex, Atta spp., Cephus spp., Diprion spp., Diprionidae, Gilpinia polytoma, Hoplocampa spp., Lasius spp., Monomorium pharaonis, Neodiprion spp., Solenopsis spp. and Vespa spp.: (l) Diptera, for example, Aedes spp., Antherigona goccata, Bibio hortulanus, Calliphora erythrocephala, Ceratitis spp., Chrysomyia spp., Cuterebra spp., Dacus spp., Delia spp., Delia radicum, Drosophila spp., e.g., Drosophila suzukii; Fannia spp., Gastrophilus spp., Glossina spp., Hypoderma spp., Hyppobosca spp., Liriomyza spp., Lucilia spp., Melanagromyza spp., Musca spp., Oestrus spp., Orseolia spp., Oscinella frit, Pegomyia hyoscyami, Phorbia spp., Rhagoletis pomonella, Sciara spp., Stomoxys spp., Tabanus spp., Tannia spp. and Tipula spp.: (m) Siphonaptera, for example, Ceratophyllus spp., and Xenopsylla cheopis; (n) from the order Thysanura, for example, Lepisma saccharina; (o) Hemiptera, for example, Bactericera sp., e.g., Bactericera cockerelli,.

The active ingredients may be applied to locations containing Scarabaeidae pests. These include but are not limited to soil, grass and various ornamental plants, trees and vegetables.

The active ingredient(s) and compositions set forth above may also be applied to locations containing the a Muscidae pest. These include but are not limited to indoor environments, garbage, animals, fences, corrals, barns, milking parlors, farrowing pens etc. containing animals (cattle, pigs, sheep, horses etc.)

The active ingredient(s) and compositions set forth above may further be applied to locations containing the active ingredient(s) and compositions containing a Tenebrionidae pest. These include but are not limited to grains, poultry and poultry dwellings enclosures (fences, corrals, barns, milking parlors, farrowing pens etc.) containing animals (cattle, pigs, sheep, horses etc.)

EXAMPLES

The composition and methods set forth above will be further illustrated in the following, non-limiting Examples. The examples are illustrative of various embodiments only and do not limit the claimed invention regarding the materials, conditions, weight ratios, process parameters and the like recited herein.

Example 1 Extraction of Chromamide, Deoxyviolacein and Violacein from Chromobacterium substugae

The following procedure is used for the purification of compounds extracted from the culture of Chromobacterium substugae:

The culture broth derived from the 10-L fermentation C. substugae in L-broth is extracted with Amberlite XAD-7 resin (Asolkar et al., 2006) by shaking the cell suspension with resin at 225 rpm for two hours at room temperature. The resin and cell mass are collected by filtration through cheesecloth and washed with DI water to remove salts. The resin, cell mass, and cheesecloth are then soaked for 2 h in acetone/methanol (50/50) after which the acetone/methanol is filtered and dried under vacuum using rotary evaporator to give the crude extract. The crude extract is then fractionated by using Sephadex LH 20 size exclusion chromatography (CH₂Cl₂/CH₃OH; 50/50) to give 7 fractions (FIG. 1). These fractious are then concentrated to dryness using rotary evaporator and the resulting dry residues are screened for biological activity using a feeding assay with Cabbage looper (Trichoplusia ni) or Beet army worm (Spodoptera exigua). The active fractions are then subjected to reversed phase HPLC (Spectra System P4000 (Thermo Scientific) to give pure compounds, which are then screened in above mentioned bioassays to locate/identify the active compounds. To confirm the identity of the compound, additional spectroscopic data such as LC/MS and NMR is recorded.

Chromamide A (1) and compound B were obtained from Fraction 1 and 2 respectively, whereas violacein (2) & deoxyviolacein (3) were purified from Fraction 5 obtained from Sephadex LH 20 chromatography.

Purification of Compounds

Purification of chromamide A (1) was performed by using HPLC C-18 column (Phenomenex, Luna 10u C18(2) 100 A, 250×10), water:acetonitrile gradient solvent system (0-10 min, 80-75% aqueous CH₃CN; 10-45 min, 75-60% aqueous CH₃CN; 45-55 min, 60-50% aqueous CH₃CN; 55-65 min. 50-100% aqueous CH₃CN; 65-70 min, 100% CH3CN; 55-70 min, 0-80% aqueous CH₃CN) at 2.5 mL/min flow rate and UV detection of 210 nm. The active compound chromamide A (I), has retention time 23.19 min.

Purification of invention compound B was performed by using HPLC C-18 column (Phenomenex, Luna 10u C18 (2) 100 A, 250×10), water:acetonitrile gradient solvent system (0-10 rain, 80-75% aqueous CH₃CN: 10-45 min, 75-60 % aqueous CH₃CN; 45-55 min, 60-50% aqueous CH₃CN; 55-65 min, 50-100% aqueous CH₃CN; 65-70 min, 100% CH₃CN; 55-70 min, 0-80% aqueous CH₃CN) at 2.5 mL/min flow rate and UV deletion of 210 nm, the active compound B, retention time 26-39 min.

Purification of violacein (2) and deoxyviolacein (3) was performed by using HPLC C-18 column (Phenomenex, Luna 10u C18(2) 100 A, 250×10), water:acetonitrile gradient solvent system (0-10 min, 70-60 V aqueous CH₃CN; 10-40 min. 60-20% aqueous CH₃CN; 40-60 min, 20-0% aqueous CH₃CN; 60-65 min. 100% CH₃CN; 65-75 min. 0-70% aqueous CH₃CN) at 2.5 mL/min flow rate and UV detection of 210 nm. the active compounds violacein (2), had a retention time 7.86 min and deoxyviolacein (3) retention time 12.45 min.

Mass Spectroscopy Analysis of Compounds

Mass spectroscopy analysis of active peaks is performed on a Thermo Finnigan LCQ Deca XP Plus electrospray (ESI) instrument using both positive and negative ionization modes in a full scan mode (m/z 100-1500 Da) on a LCQ DECA XP^(plus) Mass Spectrometer (Thermo Election Corp., San Jose, Calif.). Thermo high performance liquid chromatography (HPLC) instrument equipped with Finnigan Surveyor PDA plus detector, autosampler plus, MS pump and a 4.6 mm×100 mm Luna C18 5 μ100 A column (Phenomenex). The solvent system consisted of water (solvent A) and acetonitrile (solvent B). The mobile phase begins at 10% solvent B and is linearly increased to 100% solvent B over 20 min and then kept for 4 min. and finally returned to 10% solvent B over 3 min and kept for 3 min. The flow rate is 0.5 mL/min. The injection volume was 10 μL and the samples are kept at room temperature in an auto sampler. The compounds are analyzed by LC-MS utilizing the LC and reversed phase chromatography. Mass spectroscopy analysis of the present compounds is performed under the following conditions: The flow rate of the nitrogen gas was fixed at 30 and 15 arb for the sheath and aux/sweep gas flow rate, respectively. Electrospray ionization was performed with a spray voltage set at 5000 V and a capillary voltage at 35.0 V. The capillary temperature was set at 400° C. The data was analyzed on Xcalibur software. The chromamide A (1) has a molecular mass of 860 in positive ionization mode. The LC-MS chromatogram for another active compound B suggests a molecular mass of 874 in positive ionization mode. Violacein (2) and deoxyviolacein (3) had the molecular masses of 313 and 327 respectively in positive ionization mode.

NMR Spectroscopy Analysis of Compounds

NMR-NMR spectra were measured on a Bruker 600 MHz gradient field spectrometer. The reference is set on the internal standard tetramethylsilane (TMS, 0.00 ppm). The amino acid analyses were carried out on Hitachi 8800 amino acid analyzer.

For structure elucidation, the purified chromamide A with molecular weight 860 is further analyzed using a 600 MHz NMR instrument, and has ¹H NMR δ values at 8.89, 8.44, 8.24, 8,23, 7.96, 7.63, 6.66, 5.42, 5.36, 5.31, 5.10, 4.13, 4.07, 4.05, 3.96, 3,95, 3.88, 3.77, 3.73, 3.55, 3.44, 3.17, 2.40, 2.27, 2.11, 2.08, 2.03, 2.01, 1.97, 1.95, 1.90, 1.81, 1.68, 1.63, 1.57, 1.53, 1.48, 1.43, 1.35, 1.24, 1.07, 1.02, 0.96, 0.89. 0.88, 0.87, 0.80 (see FIG. 4) and has ¹³C NMR values of 173.62, 172.92, 172.25, 172.17, 171.66, 171.28, 170.45, 132.13, 130.04, 129.98, 129.69, 129.69, 125.48, 98.05, 70.11, 69.75, 68.30, 68.25, 64.34, 60.94, 54.54, 52.82, 49.72, 48.57, 45.68, 40.38, 39.90, 38.18, 36.60, 31.98, 31.62, 31,58, 29.53, 28.83, 27.78, 24.41, 23.06, 22.09, 20.56, 19.31, 18.78, 17.66, 15.80. The chromamide A was isolated as a white solid, which analyzed for the molecular formula C₄₃H₆₈N₆O₁₂ (13 degrees of unsaturation), by ESI high-resolution mass spectrometry (obsd M⁺ m/z 861.5376, calcd M⁺ m/z 861.5343). The ¹H NMR spectral data of chromamide A in DMSO-d₆ exhibited 68 proton signals, in which nine protons [δ_(B): 8.89, 8.44, 8.23, 8.22, 7.96, 7.64, 6.55, 5.10, 4.13], were assigned as either NH or OH due to lack of carbon correlation in a heteronuclear correlation NMR (HMQC) analysis. The ¹³C NMR spectrum, showed seven carbonyl signals [δ_(c): 173.62, 172.92, 172.25, 172.17, 171.66, 171.28, 170.45] and in the ¹H NMR spectrum, six characteristic α-amino protons signals [δ₁₁: 4.07, 4.06, 3.96, 3.95, 3.88, 3.72] were observed which demonstrate that chromamide A is a peptide.

Interpretation of 2D NMR data led to the assignment of three amino acid units of the six, one leucine (Leu), one valine (Val) and one glutamine (Gin). The presence of these amino acids were confirmed by results of amino acid analysis, which also showed the presence of the above three amino acids. Further analysis of DEFT and 2D NMR spectral data (COSY, HSQC and HMBC) established the presence three sub-structures I, 11 and III as showed below.

The connections of the three sub-structures in 1 were accomplished by routine HMBC NMR analysis using correlations between the α-amino proton and/or the secondary amide proton and the carbonyl carbon resonances and chemical shift consideration. The linkage of C-9 from sub structure 1 to C-10 from sub-structure II was established by HMBC correlations from CH₃-40 [δ₃₃: 1.00] and the α-amino proton of alanine [δ₃₁: 3.42] to the C-10 carbon [δ_(C): 70.11]. This was further confirmed by the three bond HMBC correlation from hydroxyl at [δ_(H): 5.10] to C-9 at [δ_(C): 49.78]. The methylene at [δ₁₁: 3.50] from sub-structure III showed a three bond HMBC correlation to C-19 [δ_(C): 68.31] which connected the sub structure I and II. The quaternary carbon at C-3 [δ_(C): 98 09] was connected to C-21 [δ_(C): 64.40] through a weak correlation from H-21 [δ_(H): 3.95] together with their chemical shirt values to form a one ring system. Lastly, the ring closure linkage was secured by a three-bond HMBC correlation from H₃-36 [δ_(H): 1.43] to C-1 [δ_(C)172.17], which allowed the planar structure of chromamide A (1) to be assigned.

The compound B with a molecular weight 874 exhibited similar NMR and UV data suggesting that this compound B also belongs to the class of peptide.

The structure for violacein (2) and deoxyviolacein (3) was assigned by comparison of the data of these compounds with those published in the literature. The structures of chromamide A, violacein and deoxyviolacein are shown in FIG. 2.

Example 2 Amino Acids Analysis of Chromamide A

Chromamide A (0.05 mg) was hydrolyzed by using liquid phase hydrolysis (6N HCL, 1% Phenol, 110° C., 24 hr, in vacuum). After cooling, the reaction mixture was dried and the hydrolyzed product was dissolved in Norleu dilution buffer to 1.0 mL volume. A 50 μl of the sample was loaded onto the non-exchange column for analysis.

For standards and calibration, an amino acid standards solution for protein hydrolysate on the Na-based Hitachi 8800 (Sigma, A-9906) is used to determine response factors, and thus calibrate the Hitachi 8806 analyzer for all of the amino acids. Each injection contains NorLeucine as an internal standard to allow correction of the results for variations in sample volume and chromatography variables. System utilizes Pickering Na buffers. Pierce Sequanal grade HCl (hydrolysis), a Transgenomic Ion-Exchange column and an optimized method developed by Molecular Structure Facility (MSF), UC Davis, and the individual amino acid present in the sample are reported. The amino acids present in the sample (chromamide A) were found to be Glx (Glutamine/Glutamic acid), leu (leucine) and Val (Valine).

Example 3 Effect of Chromobacterium substugae (MBI-203) Against Two Spotted Spider Mites-Bean Plants

Freeze dried MBI-203 was mixed with distilled water to varying concentrations of whole cell broth cell equivalent. Uninfested bean plants, Phaseolus vulgaris, were sprayed with MBI-203. Leaf disks were then taken from sprayed plants and placed in a petri dish as a food source for Two-Spotted Spider Mites, Tetranychus urticae. 10 mites were placed in each dish and incubated at 75° F., 12:12 (L:D), Evaluations of live and dead mites were recorded 1.3, and 7 days after infestation. 1× CFD is freeze dried material reconstituted to whole cell broth cell concentrations (0.0103 g freeze dried/mL dH₂O). The results are shown in FIG. 3.

Example 4 Effect of Chromobacterium substugae (MBI-203) Against Two Spotted Spider Mites-Marigolds

Marigolds, Tagetes erecta, were infested with Two-Spotted Spider Mites, Tetranychus urticae. Formulated product (MBI-203) or Chenopodium ambrosioides (marketed as REQUIEM® by AgraQuest, Inc., Davis, Calif.) was applied to infested plant and kept in a greenhouse with temperature ranges approximately 72-85° F. To sample, a 6 cm² leaf surface was harvested and number of live and dead nymphs and immature counted. The results are shown in Table 1.

TABLE 1 Comparison of MBI-203 with REQUIEM ® Day Day Day Day Day 0 3 5 7 14 Treatment Stage Live Live Live Live Live Untreated nymph 155 165 180 181 200 MBI-203 @ 1% nymph 146 74 144 218 216 MBI-203 @ 5% nymph 142 92.8 143 170 175 REQUIEM ® (+) nymph 142 145 147 158 153 Untreated Adult 48 60.8 75.3 77 115 MBI-203 @ 1% Adult 61.3 84.3 94 88.8 105 MBI-203 @ 5% Adult 35.5 72.3 93.8 113 111 REQUIEM ® (+) Adult 36.5 44 68 94.5 91.5

Example 5 Effect of Chromobacterium substugae (MBI-203) Screening Against two Spotted Spider Mites (TSSM)-French Beans

French beans infested with TSSM or abamectin-resistant TSSM were sprayed with 0.5%, 1%, 2%, and 4% v/v dilutions of formulated MBI-203 at approximately 100 gal/acre. Mortality was assessed 9 days after application. The results are shown in Table 2.

TABLE 2 Effect of MBI-203 on TSSM infestation in French Beans Susceptible TSSM Abamectin-resistant TSSM Reduction % Reduction % Concentration Eggs Nymphs Adults Eggs Nymphs Adults 0.5%  0 0 0 n/a n/a n/a 1% 0 0 0 0 0 0 2% 8 15 25 0 0 0 4% 55 55 60 0 0 0

Example 6 Screening MBI-203 Against two Spotted Spider Mites in Strawberry

The efficacy of five traditional compounds and MBI-203 ingredients were evaluated for TSSM control on field strawberry transplants at the Florida Gulf Coast Research and Education Center. Seedlings were transplanted in the Field (day 0), Each 12.5-ft. plot consisted of 20 plants. Plots were infested four times from day 55 through day 71 with 10 to 20 motile TSSM per plant. Seventeen treatments of various rates and schedules of application of miticides, some combined with an adjuvant, and a non-treated check were replicated four times in a RCB design. Treatments were applied using a hand held sprayer with a spray wand outfitted with a nozzle containing a 45-degree core and a number four disc. The sprayer was pressurized by CO₂, to 40 psi, and calibrated to deliver 100 gal per acre. Samples were collected weekly from day 90 before first spray through 2 wks after the last application of treatments (day 154). Samples consisted of ten randomly selected leaflets per plot and were collected from the middle one-third stratum of the plants. Motile and egg TSSM were brushed from the leaflets onto rotating sticky discs and counted. No phytotoxicity was observed. Results are shown in Tables 3 and 4.

TABLE 3 The efficacy of five traditional chemistry-derived and two biologically-derived active ingredients for TSSM control Rate Number of motile TSSM/leaflet Treatment/Formulation^(a) amt/acre day 90 day 98 day 105 day 111 day 118 day 125 Non-treated — 8.8 a 2.0 a 7.3 a 4.8 a 14.3 a 47.5 a Abamtectin as a.i^(d) . (marketed as AGRI-MEK ® 16 fl. oz. 8.5 a 4.5 a 6.3 a 5.3 a 0.5 e 0.5 h 0.15 EC, Syngenta Inc.) Bifenzate as a.i^(d) . (marketed as ACRAMITE ® 1 lb 3.8 a 6.0 a 6.0 d 0.8 c 8.3 b-f 20.5 c-e 50WS, Chemtura) + INDUCE ®^(b) COHERE ® 16 fl. oz. 2.8 a 5.5 a 7.0 a 5.5 a 14.3 ab 34.3 ab Spiromesifin as a.i^(d) + INDUCE ®^(b) 12 fl. oz. 2.8 a 2.3 a 2.0 a 2.3 a 0.5 e 0.8 gh Fenpyroximate as a.i^(d) (marketed as PORTAL ® 32 fl. oz. 4.5 a 3.8 a 6.5 a 2.8 a 2.0 c-e 7.3 c-f 5% EC, Nichino America) Hexythiazox as a.i^(d) (marketed as SAVEY ® 6 oz. 2.0 a 1.8 a 2.8 a 6.0 a 13.8 ab 23.0 ab 50DF. Gowan) MBI-203 + INDUCE ®^(b) 1 gal. 4.3 a — 9.5 a 7.8 a 10.3 a-d 18.0 a-d MBI-203 + INDUCE ®^(b) 3 gal. 2.8 a — 4.5 a 5.3 a 16.0 ab 8.8 b-e F_(17.51) 1.35 0.75 1.34 0.94 4.44 7.82 (F_(13.39)) P-value 0.20 0.70 0.21 0.53 <0.0001 <0.0001 Number of motile TSSM/leaflet Treatment/Formulation^(a) day 132 day 139 day 146 day 154 Non-treated 89.8 a 150.5 a 277.5 a 319.0 a Abamectin as a.i^(d) . (marketed as AGRI-MEK ® 3.0 d-f 6.3 fg 6.8 g-i 15.3 e-g 0.15 EC, Syngenta Inc.) Bifenzate as a.i^(d) . (marketed as ACRAMITE ® 3.0 i 28.0 ef 66.5 de 128.3 b-f 50WS, Chemtura) + INDUCE ®^(b) COHERE ® 72.5 ab 121.3 ab 237.8 a 270.0 a Spiromesifin as a.i^(d) + INDUCE ®^(b) 0.5 ef 2.5 gh 2.8 i 6.0 g Fenpyroximate as a.i^(d) (marketed as PORTAL ® 5.3 de 10.0 e-g 34.3 d-f 145.3 a-c 5% EC, Nichino America) Hexythiazox as a.i^(d) (marketed as SAVEY ® 57.5 ab 103.3 ab 196.0 ab 285.5 a 50DF. Gowan) MBI-203 + INDUCE ®^(b) 54.5 ab 97.8 a-c 196.3 ab 218.0 ab MBI-203 + INDUCE ®^(b) 22.5 bc 38.8 b-d 87.5 b-d 156.0 a-c F_(17.51) 11.12 13.26 15.86 13.84 P-value <0.0001 <0.0001 <0.0001 <0.0001 Data were transformed log₁₀ (x + 1) prior to ANOVA; non-transformed means are reported. Means within a column followed by the same letter are not significantly different by Fisher's Protected LSD (P ≦ 0.05). ^(a)A ‘+’ sign indicates the products were combined. ^(b)INDUCE ®, (Aquatic Ecosystems, Inc.), non-ionic surfactant, applied at 32 fl. oz./acre. ^(c)COHERE ®, (Helena Chemical Company), non-ionic spreader-sticker adjuvant, applied at 16 fl. oz./acre. ^(d)a.i. is active ingredient

TABLE 4 The efficacy of five traditional chemistry-derived and two biologically-derived active ingredients for TSSM control Rate Number of eggs TSSM/leaflet Treatment/Formulation^(a) amt/acre day 90 day 98 day 105 day 111 day 118 day 125 Non-treated — 15.5 a 10.0 a 27.5 ab 12.3 a 25.3 a 122.0 a Abamectin as a.i^(d) . (marketed as AGRI-MEK ® 16 fl. oz. 6.8 a 17.0 a 26.5 ab 12.0 ab 1.3 ef 2.3 gh 0.15 EC, Syngenta Inc.) Bifenzate as a.i^(d) . (marketed as ACRAMITE ® 1 lb 3.8 a 6.0 a 6.0 d 0.8 c 8.3 b-f 20.5 c-e 50WS. Chemtura) + INDUCE ®^(b) COHERE ® 16 fl. oz. 6.8 a 15.5 a 11.8 a-d 16.8 a 31.5 ab 148.3 a Spiromesifin as a.i^(d) + INDUCE ®^(b) 12 fl. oz. 7.3 a 5.3 a 7.3 d 5.0 bc 1.5 d-f 2.3 f-h Fenpyroximate as a.i^(d) (marketed as PORTAL ® 32 fl. oz. 9.3 a 11.8 a 12.3 c-d 12.5 ab 1.0 f 34.5 bc 5% EC, Nichino America) Hexythiazox as a.i^(d) (marketed as SAVEY ® 6 oz. 2.0 a 8.5 a 15.0 a-d 17.0 a 22.0 ab 71.5 ab 50DF. Gowan) MBI-203 + INDUCE ®^(b) 1 gal. 8.0 a — 27.3 ab 20.5 a 19.5 ab 56.3 ab MBI-203 + INDUCE ®^(b) 3 gal. 5.0 a — 11.0 b-d 14.5 a 11.0 a-c 30.0 bc F_(17.51) 0.95 0.96 2.12 1.93 4.48 13.52 (F_(13.39)) P-value 0.5194 0.5042 0.0201 0.0363 <0.0001 <0.0001 Number of eggs TSSM/leaflet Treatment/Formulation^(a) day 132 day 139 day 146 day 154 Non-treated 181.5 a 587.0 a 559.0 a 423.0 a Abamectin as a.i^(d) . (marketed as AGRI-MEK ® 6.5 hi 3.5 f-h 3.5 gh 41.3 e-g 0.15 EC, Syngenta Inc.) Bifenzate as a.i^(d) . (marketed as ACRAMITE ® 3.01 i 28.0 ef 66.5 de 128.3 b-f 50WS. Chemtura) + INDUCE ®^(b) COHERE ® 114.0 ab 321.0 ab 525.3 a 377.0 a Spiromesifin as a.i^(d) + INDUCE ®^(b) 2.8 hi 0.8 h 4.5 f-h 25.5 fg Fenpyroximate as a.i^(d) (marketed as PORTAL ® 20.8 de 28.5 d 97.3 cd 240.8 a-d 5% EC, Nichino America) Hexythiazox as a.i^(d) (marketed as SAVEY ® 128.5 ab 281.5 ab 393.5 ab 533.0 a 50DF. Gowan) MBI-203 + INDUCE ®^(b) 91.8 a-c 187.3 bc 304.5 ab 432.3 a MBI-203 + INDUCE ®^(b) 35.5 c-e 57.8 cd 238.3 a-c 341.5 ab F_(17.51) 18.40 19.96 19.16 5.13 P-value <0.0001 <0.0001 <0.0001 <0.0001 Data were transformed log₁₀ (x + 1) prior to ANOVA; non-transformed means are reported. Means within a column followed by the same letter are not significantly different by Fisher's Protected LSD (P ≦ 0.05). ^(a)A ‘+’ sign indicates the products were combined. ^(b)INDUCE ®, (Aquatic Ecosystems, Inc.), non-ionic surfactant, applied at 32 fl. oz./acre. ^(c)COHERE ®, (Helena Chemical Company), non-ionic spreader-sticker adjuvant, applied at 16 fl. oz./acre. ^(d)a.i. is active ingredient

Example 7 Effect of Chromobacterium substugae (MBI-203) Against House files

Test substance is screened for direct contact efficacy against houseflies (adults). There are three treatment groups for each compound: 1.5%, 3% and 6% concentration plus an untreated control. Each group contains five replicates with approximately 10 insects each. Arthropods will be treated with a hand pump sprayer until “full coverage” is reached, in 16 oz. drink caps with a beverage lid. At four hours, a cotton ball with 10% sucrose solution is provided for the flies by inserting it into the perforation in the lid. Data is taken at 5, 15, 30, 45, 60 minutes and 2, 4, and 24 hours or until endpoint. Knockdown and mortality were determined by counting the relative number of prone insects. Moribund insects were not included in mortality totals. Results are shown below in Table 5.

Example 8 Effect of Chromobacterium substugae (MBI-203) Against Utter Beetles

Three treatment groups were tested for each compound: 1.5%. 3% and 6% concentration plus an untreated control. Each group contained five replicates with approximately 10 insects each. Arthropods were treated with a hand pump sprayer until “full coverage” was reached, in 8 or 16 oz. deli squat cups with perforated lids with filter paper on the bottom to absorb any excess material. Knockdown and mortality were observed at 5, 15, 30, 45, 60 minutes and 2, 4, 24, 48, and 72 hours after treatment. Knockdown and mortality were determined by counting the relative number of prone insects. Moribund insects were not included in mortality totals. Results are shown below in Table 6.

TABLE 5 Knockdown Mortality 5 15 30 45 1 2 4 24 48 72 Rate min min min min hr hr hr hr hr. hr House 1.5%  10.0% 28.0% 54.0% 58.0% 64.0% 70.0% 76.0% 80.0% 80.0% 80.0% Fly (ZCS) 3% 14.9% 36.2% 59.6% 74.5% 80.9% 80.9% 80.9% 85.1% 89.4% 95.7% Musca 6% 47.7% 68.2% 97.7% 97.7% 97.7% 97.7% 97.7% 97.7% 100.0% 100.0% domestica

TABLE 6 Knockdown Mortality 5 15 30 45 1 2 4 24 48 72 Rate min min min min hr hr hr hr hr. hr Litter Beetle - 3% 33.3% 47.9% 50.0% 58.3% 68.8% 75.0% 77.1% 85.4% 93.8% 85.4% larvea 6% 45.1% 64.7% 72.5% 76.5% 76.5% 82.4% 76.5% 86.3% 88.2% 86.3% Alphitobius 1.5%  40.0% 58.0% 62.0% 64.0% 74.0% 78.0% 78.0% 72.0% 90.0% 86.0% diaperinus 3% 70.0% 82.0% 92.0% 86.0% 88.0% 92.0% 88.0% 94.0% 98.0% 100.0% 6% 84.3% 96.1% 98.0% 98.0% 100.0% 100.0% 100.0% 98.0% 100.0% 96.1% Litter Beetle - 1.5%  0.0% 12.0% 20.0% 22.0% 34.0% 42.0% 50.0% 68.0% 70.0% 70.0% adult 3% 2.0% 2.0% 12.0% 14.0% 24.0% 28.0% 28.0% 54.0% 54.0% 56.0% Alphitobius 6% 0.0% 8.0% 12.0% 22.0% 36.0% 40.0% 40.0% 76.0% 80.0% 78.0% diaperinus 1.5%  4.0% 6.0% 10.0% 12.0% 14.0% 18.0% 22.0% 24.0% 30.0% 32.0% 3% 6.0% 24.0% 28.0% 32.0% 42.0% 64.0% 72.0% 94.0% 94.0% 94.0% 6% 10.0% 22.0% 30.0% 38.0% 62.0% 78.0% 86.0% 86.0% 94.0% 90.0%

Example 9 Effect of MBI-203 on Egg Laying Capacity of Potato Psyllids, Bactericera cockerelli Methods

The egg laying capacity of potato psyllid females exposed to MBI-203 treated pepper leaves were determined. Pepper leaves were excised at the petiole and treated with MBI-203 by dipping for 1 minute. Treatments in the experiment were as follows: MBI-203 at 10% v/v in dH₂O, dH₂O as negative control and Avid at 10% v/v as positive control. Treated leaves were held in plastic petri dishes with the rims welled off, lined with craft foam with the center diameter cut out to expose treated leaf.

Four, 1-day old females were placed in the center of the dish where tbe treated leaf was exposed (cut out portion of the craft foam), covered with the petri plate cover, and the setup was secured with 2 binder clips. The female adults were allowed to lay eggs and egg count was done 3 to 10 days post exposure.

A follow up leaf disc bioassay on MBI-203 treated and untreated leaves was conducted to verify the effect of MBI-203 on the egg laying capacity of potato psyllid females. Treatment of MBI-203 at 3% v/v in water and an untreated control, dH₂O only (negative control) were used in the assay. Pepper leaf discs (from 3-4 week old pepper plants) were cut in circles using a 23 mm cookie cutter, selecting a flat portion of the leaf to make sure the disc can be evenly laid flat on the agar plate after treatment with the compound. The bottom of the plates was covered with 30 μL of 1% agar solution. Just enough to cover the bottom of the plate to prop leaf discs and maintain humidity. The agar was allowed to solidify by cooling at room temperature. Treatment of leaf disc was performed by pouring the treatment solution into a glass petri dish. With the solution in the dish, leaf discs were treated by soaking, swirling the dish gently to completely soak and coat the leaf discs. Treatment was performed for 1 minute and treated leaf discs were then allowed to dry for 10-15 minutes in fume hood or until the solution had completely dried off. In each solidified agar plate, 20-30 μL dH₂O was pipeted onto the agar. Each treated leaf disc was laid individually onto the agar plate, placing the leaf disc abaxial side down on wetted agar, pressed down gently to completely flatten the disc into the agar. In each plate with treated leaf disc (treatment), 4 female psyllids were introduced. The petri plates with gravid females were then covered with the petri plate cover, poked with tiny holes for aeration and to prevent condensation. The dishes were sealed with parafilm and kept at room temperature. The number of eggs laid was counted daily, starting a day post introduction of females. The experiment was done in 3 replications, repeated two times.

Results

A significant reduction of eggs by females exposed to MBI-203 treatment was observed. A slight delay in egg oviposition was apparent on females in MBI-203 treated leaf discs. Psyllid females exposed to MBI-203 treated leaf dues started ovipositing eggs 3 days post exposure (FIG. 4). Eggs laid by females peaked on day 7 and declined at day 10. At day 10, mean egg count decreased as eggs laid by females started hatching. The females exposed to the positive control treatment (Avid 10% v/v) were all dead in day 3 with no eggs deposited on the avid treated leaf discs.

Verification leaf disc bioassays confirmed a consistent result with a significant reduction of eggs oviposited by females on MBI-203 treated leaf disc at 3% v/v (FIG. 5). A 65% egg reduction was exhibited by females exposed to MBI-203 treated leaf discs compared to females exposed to untreated leaf discs (dH₂O only). These bioassay results indicate that MBI-203 affects the physiology of the psyllid females affecting their egg laying capacity.

Example 10 Effect of MBI-203 on Grubs and Scarabs Control of White Grubs on Turf Grasses

Insecticides were evaluated for control of white grubs (Southern Masked Chafer, Cyclocephala lurida Bland) on a Kentucky bluegrass (Poa pratensis L.) and perennial ryegrass rough (Lolium perenne L.) at the North Bend golf course in North Bend, Neb.. Insecticides were applied to 5×5 ft plots arranged in a randomized complete block (RCB) design with 5 replications. Liquid products were applied using a CO₂ sprayer at 40 psi and applying 174 gpa finished spray. Within 24 h following application, all treatments were irrigated with 0.25 in of water. Formulations were evaluated 24 days and 48 days after treatment (DAT) by removing from each plot three, 8-inch diameter turf-soil cores (1.05 ft² total area) to a depth of 3 inches and counting the number of surviving and moribund grubs. Plots were periodically assessed for phytotoxicity. The results are shown in Tables 7 and 8.

There appears to be a correlation between application rate and percent control for the MBI-203 DF1 treatments. All treatments, except MBI-203 DF1 (2 fl oz/100 ft2), outperformed trichlorfon 6% (marketed as DYLOX® 420 SL (6.9 fl.oz/1000 ft²) by Bayer CropScience, Inc., an industry standard insecticide for white grub control). Interestingly, moribund individuals were found in all treatments of MBI-203 AF1 and MBI-203 DF1. These numbers (in parenthesis) were not used in the statistical analysis but were included for comparative purposes. No phytotoxicity was observed. AF1 is an aqueous flowable and DF1 is a wettable powder formulation of Chromobacterium substugae.

TABLE 7 Efficacy MBI-203 in controlling White Grubs 24 days after Treatment (24 DAT) Mean Treatment/ Rate No. WG/1.05 ft² WG ± SE % formulation fl oz/1000 ft² Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 1.05 ft² Control MBI-203 AF1 16 5 (2) 4 (2) 1 2 0 2.4 ± 0.9 76.0 MBI-203 DF1 8 8 (1) 4 (1) 2 3 2 3.8 ± 1.1 62.0 MBI-203 AF1 8 3 (3) 9 (3) 7 (1) 5 (3) 0 4.8 ± 1.6 52.0 MBI-203 AF1 32 9 (3) 5 7 4 0 5.0 ± 1.5 50.0 MBI-203 DF1 4 5 6 (1) 10 2 (1) 2 (2) 5.0 ± 3.2 50.0 Dylox 420 SL 6.9 0 6 9 9 4 5.6 ± 1.7 44.0 MBI-203 DF1 2 2 (1) 8 (2) 8 9 6 6.6 ± 1.2 34.0 UTC — 9 8 15 8 10 10.0 ± 1.3  —

TABLE 8 Efficacy MBI-203 in controlling White Grubs 24 days after Treatment (43 DAT) Treatment/ Rate No. WG/1.05 ft² Mean WG % Formulation fl oz/1000 ft² Rep 1 Rep 2 Rep 3 Rep 4 1.05 ft² Control MBI-203 DF2 4.0 2 0 0 0 0.5 a 95.0 Dylox 6.2 G 3 lb 3 3 0 0 1.5 a 85.0 MBI-203 DF2 2.0 0 0 6 4 2.5 a 75.0 MBI-203 DF2 8.0 10 2 6 1 4.75 a 52.5 UTC — 15 8 10 7 10.0 b — Means followed by the same letter are not significantly different (P > 0.05, LSD = 4.4).

Feeding Study of the Effect of MBI-203 on Other Root Feeding Scarabs

Unsterilized Groton soil, 4.25 pH. 14% organic matter was infested with the scarab, oriental beetle larvae (Anomala orientalis) and rated on the number of larvae that died. The soil was dosed with aqueous flowable formulation of MBI-203 and the percent mortality computed:

TABLE 9 Effect of MBI-203 on Oriental Beetle Larvae Trial 1 Trial 2 % mortality 7 DAT (days after treatment) 1.5 ml product/5 g soil (30% soil mixture) 100 100 1 ml product/5 g soil (20% soil mixture) 100 100 .5 ml product + .5 ml de-ionized water/5 g soil 87 100 .25 ml product + .75 ml de-ionized water/5 g soil 93 100 .1 ml product + .9 ml de-ionized water/5 g soil 40 13 .05 ml product + .95 ml de-ionized water/5 g soil 27 27 Untreated Control (30% moisture) 0 7 Untreated Control (20% moisture) 0 0

A similar trial was set up with the scarab, Rhizotrogus majalis, (European chafer) grubs using 1.5 ml and 1 ml product in 5 g soil. 100/100 larvae were killed at 7 DAT and none were killed in the Control.

Trials were also conducted with black vine weevil larvae Otiorhynchus sulcatus (Curculionidae) with potting media (no food source), carrots and Taxus roots. Results are shown in Tables 10, 11, 12 and 15.

TABLE 10 Effect of MBI-203 with Potting Soil % mortality 7 DAT % mortality 14 DAT .9 ml product/3 g media 0 0 (30% soil moisture) .6 ml product/3 g media 0 0 (20% soil moisture) Control (30% moisture) 0 0 Control (20% moisture) 0 0

TABLE 11 Effect of MBI-203 with Carrots Dipped in Product % mortality 3 DAT Carrot slice dipped in product 100 Carrot slice dipped in de-ionized water (control) 0

TABLE 12 Effect of MBI-203 with Taxus Roots % mortality 3 DAT Taxus roots dipped in product and dried 90 Taxus roots dipped in de-ionized water and dried 0

TABLE 13 Effect of MBI-203 with Carrot Slice (Dried) % mortality 3 DAT Carrot slice dipped in product and dried 60 Carrot slice dipped in de-ionized water 0 and dried (control)

It appears that MBI-203 is very active against roof feeding scarab beetles and weevil, particularly when fed a treated food source.

Example 11 Effect of MBI-203 Against Cubage Root Maggot

This study was conducted to determine the efficacy of MBI-203 formulations for control of Cabbage Maggots (Delia radicum) on broccoli plants in a caged greenhouse study. Experimental treatments of MBI-203 DF-1 (a wet table powder formulation of MBI-203) at rates of 2 oz/1000 ft² and 8 oz/1000 ft²; MBI-203 DF-2 (a second wet table powder formulation of MBI-203) at rates of 2 oz/1000 ft² and 8 oz/1000 ft². Experimental treatments were compared to the commercial standard, RADIANT® (marketed by DowAgro Sciences and containing spineforam as an active ingredient) at a rate of 1 lb/gal.

The number of live adult insects was recorded weekly up to 14 days after the third application (14DA-C) and the number of live larvae was recorded weekly up to 21 DA-C. Results showed that MBI 203 DF-1 had a significantly fewer number of adults emerge by the last evaluation date, and was computable to RADIANT®. MBI-203 DF-1 had a significantly lower number of adults emerge than the UTC by the last evaluation date, and was comparable in control to Radiant.

TABLE 14 Maggot Count. Average number of larval Delia radicum insects per treatment, listed by evaluation date. Trt Treatment Rate Appl No. Name Rate Unit Code 7 DA-C 14 DA-C 21 DA-C 1 Untreated ABC 5.75 a 6.25 a 6.50 a 2 MBI 203 DF -1 2 oz/1000 ft² ABC 5.00 ab 5.00 ab 4.25 b 3 MBI 203 DF -1 8 oz/1000 ft² ABC 4.75 ab 3.00 c 3.50 bc 4 MBI 203 DF -2 2 oz/1000 ft² ABC 6.50 a 5.50 ab 4.25 b 5 MBI 203 DF -2 8 oz/1000 ft² ABC 5.25 ab 3.00 c 1.50 cd 8 Radiant 10 fl oz/a AC 1.50 c 1.00 d 1.00 d

TABLE 15 Percent Control. Average percent control of larval Delia radicum insects per treatment, listed by evaluation date. Trt Treatment Rate Appl No. Name Rate Unit Code 7 DA-C 14 DA-C 21 DA-C 1 Untreated ABC 0.00 c 0.00 d 0.00 d 2 MBI 203 DF -1 2 oz/1000 ft² ABC 13.04 bc 20.00 cd 34.62 c 3 MBI 203 DF -1 8 oz/1000 ft² ABC 17.39 bc 52.00 b 46.15 bc 4 MBI 203 DF -2 2 oz/1000 ft² ABC 0.00 c 12.00 cd 34.62 c 5 MBI 203 DF -2 8 oz/1000 ft² ABC 8.70 bc 52.00 b 76.92 ab 8 Radiant 10 fl oz/a AC 73.91 a 84.00 a 84.62 a

Example 12 Efficacy of MBI-203 DF1 and MBI-203 DF2 for the Control of Drosophila suzukii (Spotted Wing Drosophila (SWD)) on Strawberry in the Greenhouse

This study was conducted to determine the efficacy of MBI-203 DF1 and MBI-203 DF2 for the control of Spotted Winged Drosophila (SWD) on strawberry crops in the greenhouse. Experimental treatments of MBI-203 DF1 and MBI-203 DF2 were applied at rates of 1 lb/a and 4 lb/a to replicate plots. Treatments were compared to the commercial standard. Entrust® at a rate of 1.5 oz/a. All treatments were combined with the surfactant SILWET®L77 (Chemtura AgroSolutions, Inc.) at a rate of 0.05% v/v. Each replicate plot containing one strawberry plant was caged to prevent the migration of inject populations.

Third generation laboratory reared SWD winged adults were released on each caged strawberry transplant. Adult SWD counts were recorded at before application (pre-count), 4 days after application (DAA), 7 DAA, and 11 DAA. The number of SWD larvae per berry was recorded at 14 DAA, 21 DAA, 28 DAA, and 35 DAA. Statistics were analyzed using ANOVA mean comparison with LSD test and u=0.05.

Following the first application, MBI-203 treatment showed progressive reduction of Spotted Winged Drosophila adults and a rate response was observed for both DF1 and DF2 products. Although not significantly comparable to ENTRUST®, (Dow AgroBioSciences) containing spinosad as an arrive ingredient. MBI-203 DF2 at 4 lb/a significantly reduced adult populations by 25% in comparison to the UTC at 7 days after the first application (DAA). By 11 DAA, both DF1 and DF2 at 4 lb/a reduced adult counts by 44%, although not statistically different from the UTC. Both MBI-203 products exhibited significant results for reduction of SWD larvae counts. At all evaluations, DF2 at 4 lb/a significantly reduced the number of larvae per berry by 71%, comparable to ENTRUST®, (Dow AgroBioSciences) containing spinosad as an active ingredient, at 78%; further. by 21 DAA, larvae counts were controlled by all MBI-203 treatments up to 72%, at all were comparable to ENTRUST®, (Dow AgroBioSciences). A rate response was observed for both MBI-203 DF1 and DF2 with respect to larvae counts.

Results:

TABLE 16 Number of Flies per Tent. Average Count of Drosophila suzukii winged adults following release onto plants and applications per treatment, listed by evaluation date. Trt Treatment Rate Appl No. Name Rate Unit Code Pre-Count 4 DAA 7 DAA 11 DAA 1 Untreated ABC 25.00 a 23.25 a 16.00 ab 4.00 a 2 MBI-203 DF 1 1 lb/a ABC 25.00 a 21.25 a 14.75 ab 2.75 a Silwet L77 0.05 % v/v ABC 3 MBI-203 DF 1 4 lb/a ABC 25.00 a 19.75 a 13.50 ab 2.25 a Silwet L77 0.05 % v/v ABC 4 MBI-203 DF 2 1 lb/a ABC 25.00 a 21.00 a 18.00 a 3.00 a Silwet L77 0.05 % v/v ABC 5 MBI-203 DF 2 4 lb/a ABC 25.00 a 19.75 a 12.00 b 2.25 a Silwet L77 0.05 % v/v ABC 6 Entrust 1.5 oz/a ABC 25.00 a 8.25 b 0.00 c 0.00 b Silwet L77 0.05 % v/v ABC

TABLE 17 Percent Control. Average percent control of Drosophila suzukii larvae per replicate plot, listed by evaluation date. Trt Treatment Rate Appl No. Name Rate Unit Code 14 DAA 21 DAA 28 DAA 35 DAA 1 Untreated ABC 0.00 d 0.00 b 0.00 b 0.00 c 2 MBI-203 DF 1 1 lb/a ABC 41.52 c 54.22 a 42.83 ab 44.44 b Silwet L77 0.05 % v/v ABC 3 MBI-203 DF 1 4 lb/a ABC 54.78 bc 67.85 a 88.11 a 100.00 a Silwet L77 0.05 % v/v ABC 4 MBI-203 DF 2 1 lb/a ABC 52.97 bc 60.58 a 64.33 a 88.89 a Silwet L77 0.05 % v/v ABC 5 MBI-203 DF 2 4 lb/a ABC 71.36 ab 72.16 a 88.11 a 100.00 a Silwet L77 0.05 % v/v ABC 6 Entrust 1.5 oz/a ABC 78.12 a 69.19 a 100.00 a 100.00 a Silwet L77 0.05 % v/v ABC

The Findings may be Summarized as Follows:

-   -   A rate response was observed for MBI-203 DF1 and DF2 for         reduction of SWD adults and larvae counts     -   MBI-203 DF2 at 4 lb/a significantly reduced adult populations by         25% at 7 DAA     -   Throughout the trial, DF2 at 4 lb/a significantly reduced the         number of larvae per berry and was comparable to Entrust         Although application of both products did not show much effect         on adult population, it significantly reduced the larval         population in the next generation.

Example 13 Repellent Effect of MBI-203 to Aphids

Evaluation of repellent effect of various concentrations MBI-203 for green peach aphids was performed. Specifically, three treatment concentrations of MBI-203 in water (1% v/v. 3% v/v and 10% v/v) were evaluated. The MBI-203 10% v/v concentration was used as positive control and dH₂O only treatment as negative control. Each treatment solution was added with 0.01% TWEEN 20.

Bioassays were performed by treating pepper leaf discs with respective MBI-203 concentrations as mentioned above. Pepper leaf discs (from 3-4 week old pepper plants) were cut in circles using a 23mm cookie cutter, selecting a flat portion of the leaf to make sure the leaf disc can be evenly laid flat into the agar plate after treatment with the compound. A 1% agar solution was melted by heating and poured into the 145 mm×20 mm petri plate, enough to cover the bottom surface of the plate to prop leaf discs and maintain humidity. The agar was allowed to solidify by cooling at room temperature.

Leaf disc treatment was performed by pouring the treatment solution into a glass petri dish. With the solution in the dish, leaf discs were treated by soaking, swirling the dish gently to completely soak and coat the leaf discs. Treatment of leaf discs by soaking was done for 1 minute. Treated leaf discs were then allowed to dry off by taking them out from the solution using forceps and placing them in the fume hood for 10-15 minutes or until the solution had completely dried off in the leaf surface. Once leaf discs are dried, 40 μL water was pipeted onto the agar where leaf disc will be laid. Treated leaf discs were then laid equidistant from each other, onto the wetted surface of the agar, placing each disc abaxial side down. Each disc was pressed down gently to completely flatten into the agar, and 20 3-4 day old GPA adults were introduced at the center of the dish using a line paint brush. Plates were then covered and sealed with parafilm. Petri plate cover was poked with tiny holes for aeration and to prevent condensation.

The test was performed in three replications. Repellency data of adults and nymphs was determined 24 hours after exposure of adults in the plates with treated leaf discs. The number of aphids (adults and nymphs) present in each leaf was counted and data recorded and analyzed.

Results

MBI-203 Is repellent to GPA adults and nymphs with 97-99% repellency at different MBI-203 concentrations (Table 18). FIG. 6 shows a statistical difference among treatment concentrations. MBI-203 at 3% and 1% v/v concentrations had a computed mean % repellency of 97% and 99% repellency, respectively.

TABLE 18 Mean percent (%) repellency of Green Peach Aphid (GPA) adults and nymphs on pepper leaf discs treated with different MBI-203 concentrations. Treatment % Mean Repellency MBI-203 std 3% v/v 97 MBI-203 std 1% v/v 99 MBI-203 std 10% v/v 97

Example 14 MBI-203 Application Reduces Aphid Progeny

MBI-203 at 3% v/v concentration was tested to determine the effect of the compound on the progeny production of green peach aphid (GPA) adults. Bioassays were performed by iteming pepper leaf discs with MBI-203 at 3% v/v. Pepper leaf discs (from 3-4 week old pepper plants) were cut in circles using a 23 mm cookie cutter, selecting a flat portion of tbe leaf to make sure the disc can be evenly laid flat on the agar plate. A 1% agar solution was melted by heating and 30 μL, poured in each petri plate (16 mm×35 mm vented, polystyrene petri plates), just enough to cover the bottom of the plate to prop leaf discs and maintain humidity. The agar was allowed to solidify by cooling at room temperature. Leaf disc treatment was performed by pouring the treatment solution into a glass petri dish. With the solution in the dish, leaf discs were treated by soaking, gently swirling the dish to completely soak and coat the leaf discs. Treatment of leaf discs by soaking was done for 1 minute. Treated leaf discs were allowed to dry for 10-15 minutes in fume hood or until the solution had completely dried off the leaf surface. Each treated leaf disc was laid individually onto the agar plate, placing the leaf disc abaxial side down on wetted agar, pressed down gently to completely flatten the disc into the agar. In each plate with treated leaf disc (treatment). 6 GPA adults (3-4 day old) were introduced. The plates with adult aphids were then covered with parafilm. Parafilm covers were poked with tiny holes for aeration and to prevent condensation. Plates were incubated at room temperature. Progeny (early nymphal instars) were counted 3 days after adult exposure to treated leaf discs. The experiment was done in 3 replications, and the whole experiment was repeated 5 times.

FIG. 7 shows the MBI-203 significantly affected progeny production of GPA adults 3 days post exposure to treated leaf discs. The graph in figure five is the result of the five trials conducted and reduction in progeny of GPA adults was observed to be more than 50% compared to the negative control (water only treatment) and its performance is comparable to the positive control (10% Avid), showing statistically the same in progeny production. When the number of progeny production was tested in comparison with the formulated product DF2. there was no statistical difference between MBI-203 standard and DF2 at 3% v/v concentration (FIG. 8). A progeny (nymphs) reduction of more than 90% was exhibited in MBI-203 and DF2 treatments compared from the negative control, which showed to be significantly better than Avid at 10% v/v concentration (positive control).

Example 15 Extraction of Violacein and Oligo-(β-hydroxybutyric acid) from Chromobacterium substugae.

The following procedure was used for the purification of compounds extracted from the culture of Chromobacterium substugae:

The whole culture broth (WCB) derived from the 20-L fermentation C. substugae in L-broth was extracted by liquid—liquid extraction method using ethyl acetate. The ethyl acetate layer was separated and dried under vacuum using rotary evaporator to give the crude extract. The crude extract was then fractionated by using different solvent such as dichloromethane (DCM), ethyl acetate (EA), methanol (MeOH) and washing with mixture of solvent (WASH). These fractions were then concentrated to dryness using rotary evaporator and the resulting dry residues are screened for biological activity using different pest (insects, nematodes). The active fractions were then subjected to Sephadex LH 20 size exclusion chromatography (CH₂Cl₂/CH₃OH; 50/50) to give 10 fractions (FIG. 9). These fractions were then concentrated to dryness using rotary evaporator and the resulting dry residues (fractions) were screened for biological activity using insect repellency assay with green peach aphids, progeny production of green peach aphid (GPA) and nematicidal bioassay (M. incognita and/or M. hapla). The active fractions were then subjected to reversed phase HPLC (Spectra System P4000 (Thermo Scientific) to give pure compounds, which were then screened in above mentioned bioassays to locate/identify the active compounds. To confirm the identity of the compound, additional spectroscopic data such as LC/MS and NMR were recorded.

The potent insecticidal repellent compound was isolated from fractions F8, F9 & F10 and was identified as violacein (2). The nematicidal active compound from the main DCM fraction was identified as oligo-(β-hydroxybutyric acid) (4).

Muss Spectroscopy Analysis of Compounds

Mass spectroscopy analysis of active peak was performed on a Thermo Finnigan LCQ Deca XP Plus electrospray (ESI) instrument using both positive and negative ionization modes in a full scan mode (m/z 100-1500 Da) on a LCQ DECA XP^(plus) Mass Spectrometer (Thermo Electron Corp., San Jose, Calif.). Thermo high performance liquid chromatography (HPLC) instrument equipped with Finnigan Surveyor PDA plus detector, autosampler plus, MS pump and a 4.6 mm×100 mm Luna C18 5 μ 100A column (Phenomenex). The solvent system consisted of water (solvent A) and acetonitrile (solvent B). The mobile phase begins at 10% solvent B and is linearly increased to 100% solvent B over 20 min and then kept for 4 min. and finally returned to 10% solvent B over 3 min and kept for 3 min. The flow rate is 0.5 mL/min. The injection volume was 10 μL and the samples are kept at room temperature in an auto sampler. The compounds are analyzed by LC-MS utilizing the LC and reversed phase chromatography. Mass spectroscopy analysis of the present compounds is performed under the following conditions: The flow rate of the nitrogen gas was fixed at 30 and 15 arb for the sheath and aux-/sweep gas flow rate, respectively. Electrospray ionization was performed with a spray voltage set at 5000 V and a capillary voltage at 35.0 V. The capillary temperature was set at 400° C. The data was analyzed on Xcalibur software. The analysis of oligo-(β-hydroxybutyric acid) (I) was performed using LTQ Orbitrap XL hybrid Fourier Transform Mass Spectrometer at UC Davis Mass spectrometry facility.

NMR Spectroscopy Analysts of Compounds

NMR-NMR spectra were measured on a Bruker 600 MHz, gradient field spectrometer. The reference is set on the internal standard tetramethylsilane (TMS, 0.00 ppm).

Purification of Compounds

The dichloromethane (DCM) fraction was triturated with methanol and the white solid obtained was filtered off to give oligo-(β-hydroxybutyric acid) (I)

identification of Compounds Oligo-(β-hydroxybutyric Acid) (I)

The ¹H NMR spectrum of compound 4 exhibited signals at δ 5.22 (scxt), 2.62 (dd) and 2.53 (dd) with a relative intensities of 1 proton each. In addition to these a methyl signal at δ 1.31 as a doublet was also observed. The ¹³C NMR spectrum showed only four carbon signals at δ 169.2, 67.6, 40.9 and 19.8. The detail ID & 2D NMR analysis resulted to the partial structure of -(—O—CHCH₂CH₂—CO—-)- with a fragment mass of 86. The MALDI-TOF-ESI MS (FIG. 2) of this compound exhibited typical signal pattern as for a mixture of oligomers with molecular masses of [(n×86)+Na], corresponding the product was a mixture of Oligo-(β-hydroxybutyric acid) 1 with n=10-25. This compound has been reported to be isolated from several bacteria (Singh et al., 2011: Maskey et al., 2002; Hahn et al., 1995). Potency of this compound obtained from DCM fraction was confirmed in an in vitro assay using M. hapla which showed 75% immobility (FIG. 10).

In vitro Testing of Fractions and Pure Compound of Chromobacterium substugae

The fractions and pure compounds were dissolved in Dimethylsulfoxide (DMSO) and were tested in an in vitro 96-well plastic cell-culture plate bioassay. Around 15-20 nematodes in a 50 μl water solution were exposed to 100 μl of a 4 mg/ml of sample for a 24 hour period at 25° C. Once the incubation period was completed, results were recorded based on a visual grading of immobility of the juvenile nematodes (J2°s) in each well treated with sampler; each treatment was tested in well repetitions of four. Results were shown in FIG. 5, which shows the results of two different 96-well plate bioassays of C. substugae fractions and compound 4. Three controls were included in each trial; 1 positive (1% Avid) & 2 negative (DMSO & water). Both trials (T1) and (T2) were carried out using M. hapla nematodes.

Isolation and Identification of Compounds Responsible for Repellency

The main fractions such as DCM, EA, MeOH and WASH were tested for repellency activity using the given peach aphid (GPA) bioassay as described in detail below. The most potent repellency was observed for EA and MEOH fractions. The LCMS analysis of these fractions showed similar chemical profile, so since the yield of MeOH fraction was higher than EA fraction, the detail chemistry work was carried out using MeOH fraction. The MeOH fraction further fractionated using Sephadex LH 20 size exclusion chromatography (CH₂Cl₂/CH₃OH; 50/50) to give 10 fractions (FIG. 9). The most potent activity was observed in factions F9 and F10. Bioassay guided purification of these fractions by combination of HPLC and Sephadex LH 20 gave violacein as the compound responsible for repellent activity. Violacein was also tested in aphid progeny testing.

Example 16 Repellent Effect of MBI-203 Fractions on Green Peach Aphid Methods

Choice bioassays on Green Peach Aphid adults (GPA) were done using MBI-203 fractions and pure compound. Fractions and pure compound (violacein) obtained from the extraction of cell paste of C. substugae (MBI-203) were tested for insect efficacy through repellency bioassays. Pepper leaf discs (from 3-4 week old pepper plants) were cut in circles using a 23 mm cookie cutter, selecting a flat portion of the leaf to make sure the leaf disc can be evenly laid flat into the agar plate after treatment of the compound. One percent (%) agar in water was prepared. The 1% agar solution was melted by heating and poured into the 145 mm×20 mm petri plate, enough to cover the bottom surface of the plate to prop leaf discs and maintain humidity. The agar was allowed to solidify by cooling at room temperature.

Leaf disc treatment was done by pipetting gently 100 μL of MBI-203 extract unto the underside of the leaf disc. Treated leaf discs were then allowed to dry off by laying the disc flat unto a labeled 12-well plate cover. Once leaf discs are dried, 40 uL water was pipetted unto the agar where leaf disc will be laid. Treated leaf discs were then laid equidistant from each other, unto the wetted surface of the agar, placing each disc abaxial side down with the treated surface up. Each disc was pressed down gently to completely flatten into the agar. After laying treated leaf discs 20 3-4 day old GPA adults were introduced at the center of the dish using a fine paint brush. Plates was then covered and settled with parafilm. Petri plate covers were poked with tiny holes for aeration and prevent condensation.

All tests were done in three replications. To select the solvent for testing initial testing of the crude extract was done using methanol and acetone as a solvent. This test showed that acetone was better as a solvent. Succeeding samples testing including fractions and pure compound (violacein) were carried out using acetone as a solvent. Data for repellency of adults and nymphs was determined 24 hours post exposure of adults on treated leaf discs. The number of aphids (adults and nymphs) was counted and data recorded and analyzed, The percent repellency was computed as: % Repellency=100-{([N+A]“on petri dish”)/[N+A]“on petri dish” X100},

where A stands for adults and N stands for nymphs.

Results

Repellency tests for MBI-203 fractions started with the crude extracts. Crude extracts in methanol and acetone solvents were tested and analysis of result showed significant differences as shown in FIG. 11. Leaf discs treated with crude extract of MBI-203 in both methanol and acetone solvents resulted in a statistically significant difference in settling response of green peach aphid nymphs and adults than those of the negative control. FIGS. 11A and B showed a repellent effect of MBI-203 on the aphids. The methanol solvent however showed a repellent effect on aphids (FIG. 11A), showing no statistical difference with the MBI-203 extract and the positive control (avid 10%). The solvent acetone showed to be a good solvent to use in fractions as it did not exhibit repellency on GPA nymphs and adults, having the mean number of nymphs and adults settling in the leaf disc statistically the same with the negative control (FIG. 11B).

Fractions of MBI-203 exhibited a strong repellency on GPA nymphs and adults. Statistical difference among treatment means were observed (FIG. 12A and B). Fractionated materials EA and MeOH caused 100% repellency on nymphs and adults while Wash material 94% repellency which is not statistically different from the positive control (MBI-203 10% v/v) (Table 19).

TABLE 19 Mean percent repellency of MBI-203 fractionated samples. Fractionated Mean % material Repellency DCM 77.2 EA 100 203 10% (+C) 98.9 Wash 94 MeOH 100 203 10% (+C) 100 Furthermore, the 10 fractions obtained from MeOH fraction were tested using acetone as a solvent; the samples with pure violacein compound (F9, F10) showed high repellency effect on adults and nymphs of green peach aphids (Table 20). Only 2 non-violacein fractions showed high repellency effect (F2 and F3). The fractions F9 and F10 were further purified which gave violacein which had 300% repellency. The data revealed that violacein is the responsible compound in causing repellency to sucking insects. It appears that fractionated materials F6-F10 contained violacein with F9 and F10 containing pure violacein compound.

TABLE 20 Mean percent repellency of fractions obtained from fractionation of MeOH fraction (F1-F10). Fractionated material Mean % Repellency F1 81.5 F2 92.9 F3 95.6 F4 76.8 F5 84.3 F6 90.1 F7 96 F8 91.5 F9 100 F10 100

Example 17 Violacein Reduces Aphid Progeny

Two concentrations of pare violacein compound in acetone, 0.5 μg/mL and 1.0 μg/mL were used in the test to determine the effect of the compound on the progeny production of green peach aphid (GPA) adults. Bioassays were performed by treating pepper leaf discs with the different violacein concentrations in acetone. Pepper leaf discs (from 3-4 week old pepper plants) were cut into 23 mm diameter discs, selecting a flat portion of the leaf to make sure the disc can be evenly laid flat onto the agar plate after treatment with the compound. A 1% agar solution was prepared, melted by healing and 30 μL poured into petri dishes (16 mm×3.5 mm vented, polystyrene petriplates), just enough to cover the bottom of the plate to support leaf discs and maintain humidity. The agar was allowed to solidify by cooling at room temperature. Treatment with violacein was done by gently spreading 100 μL of the sample solution onto the leaf disc using a 200 μL pipetman. Treatments were set up in triplicate. Treated leaf discs were allowed to dry in the hood for 5-10 minutes. The positive control was 10% v/v Avid and the negative control was dH₂O acetone was used as the blank. In each solidified agar plate, 20-30 μL dH₂O was pipetted onto the agar to maintain humidity. Each treated leaf disc was laid individually onto the agar plate, placing the leaf disc abaxial side down on wetted agar, pressed down gently to completely flatten the disc onto the agar. In each plate with treated leaf disc (treatment), 6 GPA adults (3-4 day old) were introduced. The plates with adult aphids were then covered with parafilm. Cover parafilm was poked with holes aeration to prevent condensation, and was kept at room temperature. Progeny (early nymphal instars) were counted 3 days after adult exposure to treated leaf discs. The experiment was done in 3 replications, and the whole experiment was repeated 2 times.

As shown in FIG. 13, violacein at 1.0 μg/mL significantly reduced progeny production of adult aphids. A reduction of about 50% was observed compared to the negative control (water only treatment). The positive control (10% Avid) had the least number of progeny as most of the exposed adults died 3 days post exposure. Two trials were performed in this experiment, each treatment replicated three times. Both trials provided consistent results with violacein significantly affecting progeny.

Example 18 Other Violacein Producers Display Repellency Against Aphids

The repellent effect of other Chromobacterium species on green peach aphids was also evaluated. Chromobacterium species evaluated are: Chromobacterium piscinae DSM 23278, C. pseudoviolaceum DSM 23279. C. haemolyticum DSM 19808 and C. aquaticum DSM 19852. Two of the species are violacein producing species (C. piscinae and C. pseudoviol) while the two other species are documented to not produce violacein. Microorganisms were grown on LB broth at 26° C., and 100 rpm for 5 days. At the end of the fermentation, broths were harvested and aliquoted for bioassay. Treatment concentrations at 5% v/v in water were tested on GPA adults. MBI-203 10% v/v concentration was used as positive control and dH20 only treatment as negative control. Each treatment solution was added with 0.01% TWEEN 20.

Bioassays were performed by treating pepper leaf discs as previously described. Each treated disc was pressed down gently to completely flatten into the agar. After laying treated leaf discs, 20 3-4 day old GPA adults were introduced at the center of the dish using a line paint brush. Plates were then covered and scaled with parafilm. Petri plate covers were poked with tiny holes for aeration and to prevent condensation.

The test was done in three replications. Repellency data of adults and nymphs was determined 24 hours after exposure of adults into the plates with treated leaf discs. The number of aphids (adults and nymphs) settled on each leaf disc was counted and data recorded and analyzed. Percent repellency was calculated as follows:

${\% \mspace{14mu} {Repellency}} = {100 - \left\{ {\frac{\left\lbrack {N + A} \right\rbrack {on}\mspace{14mu} {treated}\mspace{14mu} {leaf}}{\left\lbrack {N + A} \right\rbrack {on}\mspace{14mu} {petri}\mspace{14mu} {dish}}100} \right\}}$

Where N=Number of Nymphs, and A=Number of Adult Aphis

Results are shown in Tables 21 and 22 and FIGS. 14 and 15. Several violacein-producing Chromobacterium species showed repellency to GPA adults and nymphs with statistical differences among treatment means. Chromobacterium species producing violacein had mean % repellency of 75% (C. piscinae), 86% (C. pseudoviolaceum), while non-violacein producers (C. aquaticum and C. haemolyticum) were not statistically different from the untreated control (water). A slight trend of repellency was observed for the non-violacein producers.

TABLE 21 Mean percent (%) repellency of Green Peach aphid (GPA) adults and nymphs exposed to pepper leaf discs treated with different Chromobacterium species. Treatment Mean % Repellency C. piscinae 75 C. pseudoviolaceum 86 MBI-203 Std 10% 100

TABLE 22 Mean percent (%) repellency of Green Peach aphid (GPA) adults and nymphs exposed to pepper leaf discs treated with different Chromobacterium species Treatment Mean % Repellency C. aquaticum 91.4 C. haemolyticum 67.3 MBI-203 Std 10% 99

Example 19 Comparison of Stability of Chromobacterium Formulation with and without Sodium Benzoate

Formulation 1 contains Chromobacterium cell concentrated harvest 32 parts, Chrombacterium Fermentation Broth Supernatant 623 parts, n-Hexanol 1 part, Sodium Alginate 0.5 parts, Sorbitan Ester Ethoxylate 2 parts, and d-limonene 2 parts. These formulation ingredients are chosen for their functionality in ensuring uniform and stable mixtures and are also preferred due to their listing on US EPA list 4. A listing of an ingredient on EPA list 4 deems that it is of minimal concern in terms of effect on the environment and toxicology. Formulation 2 contains Chromobacterium Cell concentrated harvest 32 parts, Chromobacterium Fermentation Broth Supernatant 54.5 pans. n-Hexanol 1 part. Sodium Alginate 0.5 parts. Sorbitan Ester Ethoxylate 2 parts, and Sodium Benzoate 10 parts.

Table 23 illustrates results of storage of formulations 1 & 2 over and extended period of time.

TABLE 23 Storage of Formulations 1 and 2 Visual Observation: Formulation One day 30 days 120 days Formulation 1 Uniform Separation of white Non uniform Grey purple light brown layers brown liquid with liquid on top. Reduced while crusting in purple color head space. Formulation 2 Uniform Uniform purple Uniform purple purple liquid liquid liquid

Formulation 2 is more stable than Formulation 1. It appears that water soluble salts of benzoic acid stabilizes biological pesticide compositions against physical separation and less of activity due to exposure to sunlight. The benzoate ions provide solvency and electrolyte balancing properties such that the biological matrix remains uniform this results in extended shelf life for the pesticide composition. The benzoate ions also provide ultraviolet radiation absorption for the product once it has been applied to field crops. The UV protection extends the insecticide activity for at least several more days.

Example 20 Effect of Sodium Benzoate on Pest Mortality

The end product MBI-203 containing Chromobacterium substugae, d-limonene, hexanol, propylene glycol, and paraben formulation (MBI-203 EP) combined with calcium carbonate, sodium benzoate, or titanium oxide were placed into plastic petri dishes and sealed with parafilm. Plates were placed outside for 7 hours in the sunlight. After exposure to sun, material was brought inside and diluted to 1.5 and 3% v/v concentrations with autoclaved Millipore water. Material was then placed on artificial diet, dried, and fed to neonate Cabbage Looper, Trichoplusia ni. Mortality was scored 3 and 4 days after infestation of diet. The results are shown in Table 24 and FIG. 16.

TABLE 24 Effect of Sodium Benzoate on Pest Mortality Composition Mortality (% loss) EP + CaCO₃ ~30% loss EP + TiO₂ ~60% loss EP + Purshade ~25% loss EP + Na benzoate ~10% loss EP only ~30% loss

Broccoli Study

4-5 week old Packman broccoli were sprayed with 3% v/v dilutions of MBI-203 end product (d-limonene formulation) (MBI-203 EP) with and without sodium benzoate. Each plant in a 9 in² pot received 500 μL of treatment. Tween-20 at a final concentration of 0.01% was included in all samples. Five adult Cabbage Aphids, Brevicoryne brassicae, were placed on each plant. Plants and insects ware incubated under growth lights (75-85° F., 16 h light/8 h dark). Alive Aphids were scored 3, 5, and 7 days after infestation. It appeared that the sodium benzoate formulation provided better control than the end product alone (FIG. 17).

Red Cabbage Study

10% dilutions of MBI-203 end product (d-limonene formulation) (MBI-203 EP) with and without sodium benzoate were sprayed onto Red Cabbage at approximately 30 gal/acre treatment rates. A formulation blank (lot 2403-83-3) at 10% v/v was also included in fix assay. After plants had dried, they were infested with 10 Cabbage Aphids. The assay was scored on days 3, 6, and 8. Percent control was determined by applying the Henderson-Tilton correction. The results are shown in FIG. 18. The end product+sodium benzoate formulation had better control of aphids than the end product alone or the formulation blank alone.

Example 21 Comparison of Sodium Benzoate and Calcium Carbonate

Undiluted MBI-203 end product (MBI-203 EP) with various concentrations of sodium benzoate or calcium carbonate were exposed to sunlight for 1 day in sealed plastic petri dishes. Sun-exposed and unexposed material were then diluted to 3% v/v dilutions and applied to artificial diet. Neonate Cabbage Loopers were exposed to diet and mortality was scored 4 days after infestation of diet. Results are shown in FIG. 19. Sodium benzoate at 10% and 15% concentrations appeared to provide the least degradation in activity at the most economical price.

Example 22 Effect of Lignin Sulfonate

Undiluted spray dried cells with various additives were poured into a plastic vial and exposed to the environment for 4 days. 40-65° F., sunny with some days of sprinkles). Exposed and unexposed samples were diluted to 6% v/v and sprayed onto Packman Broccoli plants. Dried plants were infested with 5 immature Cabbage Aphids. Aphids were scored 3 and 6 days after infestation. Control was determined by applying the Henderson-Tilton correction. Spray dried cells with sodium benzoate had one of the highest kills at day 3 and maintained control as the assay continued to day 6 (see FIG. 20).

MBI-203 end product (d-limonene formulation) (MBI-203 EP) was mixed with various concentrations of sodium benzoate and lignin sulfonate. Treatments were diluted to 10% v/v concentrations and pipetted into plastic petri dishes and exposed to sunlight for 4 continuous days. Samples were then tested for Cabbage Looper activity by treating artificial diet. Lignin sulfonate treatments tended to form a crust over on top of artificial diet. End product with only sodium benzoate seemed to have the most promising kill before and after sun exposure (see FIGS. 21 and 22).

In another study, 10% v/v dilutions of MBI-203 end product (d-limonene formulation) with 10% sodium benzoate and various concentrations of lignin sulfonate were exposed to sunlight for 4 consecutive days in sealed plastic petri dishes. After exposure, 4 week old Packman Broccoli were sprayed with treatments at approximately a 30 gal/acre rate. Three late second instar Cabbage Looper larvae were placed on each treated plant and mortality was scored on days 3 and 4 of the assay. Of the plants in the sun, tbe sodium benzoate only sample had the highest control (see FIG. 23).

Although this invention has been described with reference to specific embodiments, the details thereof are not to be construed as limiting, as it is obvious that one can use various equivalents, changes and modifications and still be within the scope of the present invention.

Various references are cited throughout this specification, each of which is incorporated herein by reference in its entirety.

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1-17. (canceled)
 18. A stabilized biological pesticide composition comprising: (a) a whole cell broth, filtrate, supernatant, extract or pesticidally active substance derived from Chromobacterium species fermentation with pesticidal activity; and (b) a sunlight protecting agent, wherein said sunlight protecting agent is a lignin salt and/or benzoic acid salt.
 19. The composition of claim 18, wherein said benzoic acid salt is selected from sodium, potassium, calcium, magnesium, and combinations thereof and said lignin salt is selected from lignin sulfonate salts from the neutralized ions of sodium, potassium, calcium, magnesium, and combinations thereof.
 20. The composition according to claim 18, wherein said Chromobacterium species is Chromobacterium subtsugae (NRRL B-30655).
 21. The composition of claim 18, wherein said Chromobacterium species is present in the amount of at least about 5% and said sunlight protecting agent is present in the amount of at least about 5%.
 22. A method for stabilizing a composition against physical separation and loss of pesticidal activity due to exposure to sunlight, wherein said composition comprises an isolated Chromobacterium species, wherein the method comprises the step of providing an amount of a stabilizing agent to said composition effective to stabilize the composition against physical separation and loss of pesticidal activity due to exposure to sunlight.
 23. (canceled)
 24. The method of claim 22, wherein the stabilizing agent is selected from a benzoic acid salt and a ligonsulfonate salt.
 25. The method of claim 22, wherein said Chromobacterium species is present in the amount of at least about 5% and said sunlight protecting agent is present in the amount of at least about 5%.
 26. The method of claim 22, wherein said Chromobacterium species is Chromobacterium subtsugae (NRRL B-30655). 